大鼠行为学改变与黑质多巴胺能神经元的相关性研究

目的　观察毁损黑质多巴胺能神经元大鼠行为学及其形态学变化特点 ,探讨两者之间的相关性。方法　利用 6 -羟基多巴胺 (6 - OHDA)单侧一点注射大鼠黑质致密区 (SNc) ,特异毁损多巴胺 (DA )能神经元 ,采用开野实验观察术后 1d、3d、5 d、7d、14 d、2 1d行为学变化 ;利用 Nissl染色、HE染色、免疫组织化学方法和电镜的方法 ,观察各时间点黑质形态学变化。结果　毁损侧 DA能神经元逐渐减少 ,超微结构损伤逐渐加重 ;开野实验中旋转、探究、后肢站立和穿梭行为在术后 1d即有显著改变 (与对照组比较 P<0 .0 5 ) ,其中 ,旋转行为与毁损程度呈正相关 (r=0 .4 71,P <0 .0 1) ,探究、后肢站立和穿梭行为与毁损程度呈负相关 (r分别为 - 0 .719、- 0 .5 89、- 0 .5 94 ,P <0 .0 1) ,修饰行为与毁损程度无相关性 (r=- 0 .2 2 7,P>0 .0 5 )。结论　黑质 DA能神经元丢失是毁损大鼠行为改变的病理学基础 ,开野实验可作为丢失程度的敏感行为学观察指标。     

Pharmacokinetic,neurochemical, stereological and neuropathological studies on the potential effects of paraquat in the substantia nigra pars compacta and striatum of male C57BL/6J mice

The pharmacokinetics and neurotoxicity of paraquat dichloride (PQ) were assessed following once weekly administration to C57BL/6J male mice by intraperitoneal injection for 1, 2 or 3 weeks at doses of 10, 15 or 25 mg/kg/week. Approximately 0.3% of the administered dose was taken up by the brain and was slowly eliminated, with a half-life of approximately 3 weeks. PQ did not alter the concentration of dopamine (DA), homovanillic acid (HVA) or 3,4-dihydroxyphenylacetic acid (DOPAC), or increase dopamine turnover in the striatum. There was inconsistent stereological evidence of a loss of DA neurons, as identified by chromogenic or fluorescent-tagged antibodies to tyrosine hydroxylase in the substantia nigra pars compacta (SNpc). There was no evidence that PQ induced neuronal degeneration in the SNpc or degenerating neuronal processes in the striatum, as indicated by the absence of uptake of silver stain or reduced immunolabeling of tyrosine-hydroxylase-positive (TH⁺) neurons. There was no evidence of apoptotic cell death, which was evaluated using TUNEL or caspase 3 assays. Microglia (IBA-1 immunoreactivity) and astrocytes (GFAP immunoreactivity) were not activated in PQ-treated mice 4, 8, 16, 24, 48, 96 or 168 h after 1, 2 or 3 doses of PQ.In contrast, mice dosed with the positive control substance, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 10 mg/kg/dose × 4 doses, 2 h apart), displayed significantly reduced DA and DOPAC concentrations and increased DA turnover in the striatum 7 days after dosing. The number of TH⁺ neurons in the SNpc was reduced, and there were increased numbers of degenerating neurons and neuronal processes in the SNpc and striatum. MPTP-mediated cell death was not attributed to apoptosis. MPTP activated microglia and astrocytes within 4 h of the last dose, reaching a peak within 48 h. The microglial response ended by 96 h in the SNpc, but the astrocytic response continued through 168 h in the striatum.These results bring into question previous published stereological studies that report loss of TH⁺ neurons in the SNpc of PQ-treated mice. This study also suggests that even if the reduction in TH⁺ neurons reported by others occurs in PQ-treated mice, this apparent phenotypic change is unaccompanied by neuronal cell death or by modification of dopamine levels in the striatum.     

Exogenousl-5-hydroxytryptophan is decarboxylated in neurons of the substantia nigra pars compacta and locus coeruleus of the rat

The aim of the present study is to examine by immunohistochemistry whether exogenousl-5-hydroxytryptophan (l-5HTP) is decarboxylated in neurons of the substantia nigra pars compacta (SNC) and locus coeruleus (LC) of the rat. In normal rats, neurons of the SNC and LC stained intensely for aromaticl-amino acid decarboxylase (AADC). No serotonin (5HT)-positive cells were found in the two regions of the normal rats. In rats that were intraperitoneally injected withl-5HTP alone, the SNC neurons stained deeply for 5HT, but the LC neurons showed only a faint staining for 5HT. In rats that intraperitoneally received both a monoamine oxidase (MAO) inhibitor andl-5HTP, when compared with thel-5HTP-injected rats, the LC neurons became much darker in 5HT staining, but the SNC neurons showed only a slight increase in 5HT staining. The present findings suggest that (i) AADC in dopaminergic neurons of the SNC and in noradrenergic neurons of the LC can catalyze the in vivo decarboxylation of exogenousl-5HTP to produce 5HT, and (ii) most of the newly produced 5HT in the LC neurons is rapidly degraded by endogenous MAO.     

Lesion of Subthalamic Nucleus in Parkinsonian Rats : Effects of Dopamine D1 and D2 Receptor Agonists on the Neuronal Activities of the Substantia Nigra Pars Reticulata

Objective

It was hypothesized that dopamine agonist administration and subthalamic nucleus (STN) lesion in the rat might have a synergistic effect on the neuronal activities of substantia nigra pars reticulata (SNpr) as observed in patients with Parkinson''s disease. The effects of {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 (a D₁ receptor agonist) and Quinpirole (a D₂ receptor agonist) were compared in parkinsonian rat models with 6- hydroxydopamine (6-OHDA) after STN lesion.

Methods

{"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 and Quinpirole were consecutively injected intrastriatally. SNpr was microrecorded to ascertain the activity of the basal ganglia output structure. The effect of {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 or Quinpirole injection on the firing rate and firing patterns of SNpr was investigated in medial forebrain bundle (MFB) lesioned rats and in MFB+STN lesioned rats.

Results

The administration of {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 decreased SNpr neuronal firing rates and the percentage of burst neurons in the MFB lesioned rats, but did not alter them in MFB+STN lesioned rats. The administration ofQuinpirole significantly decreased the spontaneous firing rate in the MFB lesioned rats. However, after an additional STN lesion, it increased the percentage of burst neurons.

Conclusion

This study demonstrated that dopamine agonists and STN lesion decreased the hyperactive firing rate and the percentage of burst neurons of SNpr neurons in 6-OHDA lesioned rats, respectively. Quinpirole with STN lesion increased a percentage of burst neurons. To clear the exact interactive mechanism of D₁ and D₂ agonist and the corresponding location, it should be followed a study using a nonselective dopamine agonist and D₁, D₂ selective antagonist.     

Substantia nigra pars reticulata single unit activity in normal and 60HDA-lesioned rats: Effects of intrastriatal apomorphine and subthalamic lesions

The spontaneous activity and the response to intrastriatal application of apomorphine of substantia nigra pars reticulata (SNpr) single units was studied in four experimental groups of rats: (1) normal rats; (2) subthalamic nucleus (STN) lesioned rats; (3) rats bearing a 6-hydroxydopamine (6OHDA) lesion; and (4) 6OHDA-lesioned animals with an additional STN lesion. Thirty-eight percent of units from 6OHDA-lesioned rats showed a bursting pattern of spontaneous activity, which was never found in normal rats. STN lesions had no effect on the spontaneous activity of SNpr units from normal rats, but reduced the percentage of burst units in 6OHDA-lesioned animals. Intrastriatal apomorphine produced responses in 62% of SNpr units from normal rats and 85% of units from 6OHDA-lesioned animals (P < 0.05). In addition, the modifications in the firing rate and in the coefficient of variation of the interspike intervals induced by intrastriatal apomorphine were significantly greater for the units isolated from 6OHDA-lesioned rats. In particular, it was noted that all the burst units responded to apomorphine, showing the highest changes in firing rate and coefficient of variation. However, intrastriatal apomorphine did not always turn the activity of burst units into a more physiological pattern. STN lesions reduced the percentage of units responding to intrastriatal apomorphine in normal rats. In 6OHDA-lesioned rats, STN lesions reduced the number of responsive units, and their change in mean firing rate and coefficient of variation. Our results show that the STN participates in the genesis of the bursting pattern of activity of SNpr units in 6OHDA-lesioned rats, and that STN lesions can partially revert the abnormal spontaneous and apomorphine-induced responses of SNpr units in these animals. Synapse 27:278–293, 1997. © 1997 Wiley-Liss, Inc.     

Exogenousl-5-hydroxytryptophan is decarboxylated in neurons of the substantia nigra pars compacta and locus coeruleus of the rat

The aim of the present study is to examine by immunohistochemistry whether exogenousl-5-hydroxytryptophan (l-5HTP) is decarboxylated in neurons of the substantia nigra pars compacta (SNC) and locus coeruleus (LC) of the rat. In normal rats, neurons of the SNC and LC stained intensely for aromaticl-amino acid decarboxylase (AADC). No serotonin (5HT)-positive cells were found in the two regions of the normal rats. In rats that were intraperitoneally injected withl-5HTP alone, the SNC neurons stained deeply for 5HT, but the LC neurons showed only a faint staining for 5HT. In rats that intraperitoneally received both a monoamine oxidase (MAO) inhibitor andl-5HTP, when compared with thel-5HTP-injected rats, the LC neurons became much darker in 5HT staining, but the SNC neurons showed only a slight increase in 5HT staining. The present findings suggest that (i) AADC in dopaminergic neurons of the SNC and in noradrenergic neurons of the LC can catalyze the in vivo decarboxylation of exogenousl-5HTP to produce 5HT, and (ii) most of the newly produced 5HT in the LC neurons is rapidly degraded by endogenous MAO.     

Valproic acid enhances axonal regeneration and recovery of motor function after sciatic nerve axotomy in adult rats

It has recently been demonstrated that valproic acid (VPA) robustly promotes neurite outgrowth, activates the extracellular signal regulated kinase pathway, and increases growth cone-associated protein 43 and bcl-2 levels in cultured human neuroblastoma SH-SY5Y cells. We hypothesized that VPA could also enhance peripheral nerve regeneration in adult animals. To test this hypothesis, we examined the effects of VPA (300 mg/kg daily for 16 weeks) on sciatic axonal regeneration following single or conditional axotomies in rats. The results showed that in VPA-treated rats there was a significant increase in the total numbers of regenerated myelinated nerve fibers and reinnervated muscle fibers in comparison with those rats not treated with VPA. As measured by sciatic function index and toe spread index, the motor function of the reinnervated hind limbs of rats receiving single axotomy without VPA treatment significantly improved at week 8 and reached plateau levels at about week 11, whereas the motor function of the reinnervated hind limbs of rats receiving single axotomy plus VPA and rats receiving conditional axotomy with or without VPA treatment significantly improved at week 4 and reached plateau levels at about week 8; there was no significant difference of the motor function among the three later groups. The results demonstrated that VPA is able to enhance sciatic nerve regeneration and recovery of motor function in adult rats, suggesting the potential clinical application of VPA for the treatment of peripheral nerve injury in humans.     

Evidence for Functional Differences between Entopeduncular Nucleus and Substantia Nigra: Effects of APV (DL-2-amino-5-phosphonovaleric acid) Microinfusion on Reaction Time Performance in the Rat

Overactivity of the excitatory amino acid outputs of the subthalamic nucleus (STN) has recently been found to be one of the cascade of subsequent disruptions caused by nigrostriatal dopaminergic degeneration in Parkinson's disease. The respective contribution of the excitatory glutamatergic output structures of the STN [i.e. the globus pallidus (GP), entopeduncular nucleus (EP) and substantia nigra pars reticulata (SNr)] to the control of movement is not known, however. To investigate further the function of glutamatergic transmission through NMDA receptor subtypes in these three structures, the effects of discrete local infusion of a competitive receptor antagonist, DL-2-amino-5-phosphonovaleric acid (APV), into the EP, GP and SNr were tested in rats performing a reaction time task. Bilateral infusion of APV into the different output structures of the STN differentially impaired the performance of rats trained to release a lever after the onset of a visual stimulus within a time limit to obtain a food reward. Infusion of APV (0.25 and 0.5 μg/0.5 μl) into the SNr was found to induce behavioural deficits characterized by a dramatic increase in the number of premature lever releases and decreased mean reaction time. In contrast, the infusion of APV at a dose of 0.25 μg into the GP or EP was found to induce a motor initiation deficit characterized by an increased number of delayed responses (lever release after the time limit) and increased mean reaction time. At a dose of 0.5 μg, a premature responding deficit was added to the previous motor impairment. Interestingly, when APV was infused simultaneously into the GP and SNr in the same animals, the behavioural effects tended to be similar to those observed after a single infusion into the SNr. Altogether, these results reveal that the different functional weight of the three main output pathways originating at the STN level is t.o. The behavioural deficits induced by NMDA receptor blockade in the SNr were similar to those observed previously after a neurotoxic lesion of the STN, suggesting that NMDA receptors in this structure play a major role as a functional output of the STN. Furthermore, regarding the differential effects produced by the same dose of APV in the SNr and the EP, these two structures, which are classically believed to be functionally linked should not be considered as the same functional entity in the organization of basal ganglia outflow.     

苯甲酸雌二醇对去势(癎)性发作大鼠海马基因表达的影响

目的 分析苯甲酸雌二醇（estradiol benzoate,EB）预处理的去势大鼠经红藻氨酸（kainic acid,KA）诱导痫性发作后海马基因表达的图谱,探讨雌激素对痫性发作大鼠海马的影响.方法 应用含有10 000个基因的cDNA芯片,检测EB干预对KA诱导的去势大鼠痫性发作后海马组织基因表达的影响.应用功能富集分析,筛选有统计学差异的基因功能群.结果 EB逆转了KA致痫后有显著差异表达的基因共392个,其中下调的基因258个（65.82%）,上调的基因134个（34.18%）.经功能富集分析,共筛选出8个主要功能群,其中下调的功能群5个（共21个基因）,主要涉及凋亡、抗凋亡与神经发生、长时程突触传递增强效应等;上调的功能群有3个（共4个基因）,涉及细胞膜受体相关的信号转导等. 结论 EB能逆转KA诱导的去势大鼠痫性发作后海马神经元的基因表达,且可能以促进神经元凋亡为主.     

Effect of the 21-aminosteroid U74006F and methylprednisolone on motor function recovery and oedema after spinal cord compression in rats

The effect of the 21-aminosteroid U74006F and methylprednisolone (MP) on motor function and oedema were investigated after spinal cord compression in rats. Each compound was administered i.v. as a single dose 60 min after injury. The hind limb motor function was assessed using the inclined plane technique and expressed as the capacity angle. The water content was calculated as the percent wet weight of the total weight. Prior to compression the capacity angle was close to 62–64°. One day after compression the motor function was reduced significantly in all rats. However, the capacity angle was significantly higher after treatment with U74006F or MP than with vehicle, i.e. 50°± 4, 45°± 5, and 32°± 3, respectively. This improved functional recovery persisted during the initial nine days. After compression of the spinal cord the water content increased to a maximum on day 4 in all groups. The water content was not significantly different in any of the groups except on day one and nine when it was less in groups treated with U74006F. In conclusion, a single i. v. injection of U74006 or MP given 60 min after compression of the spinal cord improved motor function without effecting oedema expressed as water content.     

The effects of lateralized training on spontaneous forelimb preference, lesion deficits, and graft-mediated functional recovery after unilateral striatal lesions in rats

The ability of striatal embryonic grafts to promote functional recovery on complex behavioral tasks depends on various factors, including the amount of striatal-like tissue within the grafts and the duration of post-graft training. However, how the innate paw bias of animals is affected by experience, or influences recovery following injury, is less known. Here, we have examined the effects of intrinsic side bias and lateralized limb use training on spontaneous forelimb preference and graft-mediated functional recovery in a skilled reaching task in a rodent model of Huntington's disease. Na?ve rats were assessed on their baseline paw preferences when reaching between the bars of their cage to retrieve sugar pellets from a tray attached outside. Next, rats were lesioned unilaterally in the lateral dorsal striatum with quinolinic acid, and 7-10 days later, half of the animals were given suspension grafts prepared from E15 whole ganglionic eminence implanted into the lesioned striatum. The animals then received extensive unilateral training, either ipsi- or contralateral to the side of the lesion and graft in separate subgroups, on the 'staircase' task until asymptotic performance was obtained. As reported previously, the grafts alleviated lesion-induced deficits in retrieving pellets from the contralateral staircase. Spontaneous biases were then reassessed in the cage-reaching task. Irrespective of whether the animal received ipsilateral or contralateral staircase training, the unilateral lesions induced a significant shift in spontaneous bias towards the ipsilateral paw. Grafted animals showed a similar shift in bias if staircase training was given to the ipsilateral paw but showed no change in spontaneous bias (similar to controls) if they had received contralateral training during the post-transplantation period. The results suggest that striatal grafts can alleviate lesion-induced changes in their spontaneous side preferences, but only if they receive extensive training in the use of the contralateral limb, compatible with the notion that recovery is use-dependent.     

Effects of p38 MAPK signaling pathway on cognitive function and recovery of neuronal function after hypoxic-ischemic brain injury in newborn rats

To explore the effects of p38 MAPK signaling pathway on cognitive function and recovery of neuronal function after hypoxic-ischemic brain injury (HIBI) in newborn rats. Seventy-two healthy SPF grade SD newborn rats were randomly and equally divided into Normal group (healthy rats) and Sham group (rats underwent sham operation), Model group (HIBI model rats), p38 MAPK Inhibitor group (HIBI model rats treated with p38 MAPK inhibitor) and p38 MAPK Activator group (HIBI model rats treated with p38 MAPK activator). On postnatal day 28, Morris water maze, tail suspension test and inclined plane test were conducted on rats in each group. Twenty-four hours after modeling, the expression of p-p38 MAPK protein and apoptosis related genes in rat hippocampal tissues was detected by TUNEL staining, qRT-PCR and Western blot. Compared with Normal group, escape latency and inclined plane test time were prolonged, the number of passing through the platform and tail suspension time were reduced (all P < 0.05); Bax and Caspase-3 mRNA and protein expression levels and p-p38 MAPK protein level were increased, Bcl-2 mRNA level was decreased, and neuronal apoptosis proportion was increased in Model group (all P < 0.05). Compared with Model group, the above indicators showed reversed and enhanced trends in p38 MAPK Inhibitor and p38 MAPK Activator groups, respectively (all P < 0.05). Inhibition of p38 MAPK signaling pathway can effectively improve the learning and memory ability and motor function of newborn rats with HIBI, and reduce neuronal apoptosis in the hippocampal tissues, thereby promoting neuronal recovery.     

Effects of low-frequency repetitive transcranial magnetic stimulation and neuromuscular electrical stimulation on upper extremity motor recovery in the early period after stroke: a preliminary study

Objective: To assess the efficacy of inhibitory repetitive transcranial magnetic stimulation (rTMS) and neuromuscular electrical stimulation (NMES) on upper extremity motor function in patients with acute/subacute ischemic stroke.

Methods: Twenty-five ischemic acute/subacute stroke subjects were enrolled in this randomized controlled trial. Experimental group 1 received low frequency (LF) rTMS to the primary motor cortex of the unaffected side + physical therapy (PT) including activities to improve strength, flexibility, transfers, posture, balance, coordination, and activities of daily living, mainly focusing on upper limb movements; experimental group 2 received the same protocol combined with NMES to hand extensor muscles; and the control group received only PT. Functional magnetic resonance imaging (fMRI) scan was used to evaluate the activation or inhibition of the affected and unaffected primary motor cortex.

Results: No adverse effect was reported. Most of the clinical outcome scores improved significantly in all groups, however no statistically significant difference was found between groups due to the small sample sizes. The highest percent improvement scores were observed in TMS + NMES group (varying between 48 and 99.3%) and the lowest scores in control group (varying between 13.1 and 28.1%). Hand motor recovery was significant in both experimental groups while it did not change in control group. Some motor cortex excitability changes were also observed in fMRI.

Conclusion: LF-rTMS with or without NMES seems to facilitate the motor recovery in the paretic hand of patients with acute/subacute ischemic stroke. TMS or the combination of TMS + NMES may be a promising additional therapy in upper limb motor training. Further studies with larger numbers of patients are needed to establish their effectiveness in upper limb motor rehabilitation of stroke.     

Dopamine-nociceptin/orphanin FQ interactions in the substantia nigra reticulata of hemiparkinsonian rats: involvement of D2/D3 receptors and impact on nigro-thalamic neurons and motor activity

Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor antagonists proved to be effective in alleviating experimental parkinsonism. Nonetheless, loss of effectiveness or even worsening of parkinsonian symptoms have been observed at high doses. With the aim of clarifying the circuitry underlying the dual action of NOP receptor antagonists and the role of endogenous dopamine, the NOP receptor antagonist 1-benzyl-N-[3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl]pyrrolidine-2-carboxamide (Compound 24) and the D₂/D₃ receptor antagonist raclopride were used in 6-hydroxydopamine hemilesioned rats. Systemically administered Compound 24 improved motor activity in the 0.1–10 mg/kg dose range being ineffective at 30 mg/kg. To confirm NOP selectivity, Compound 24 improved motor performance in wild-type mice at 1 and 10 mg/kg and inhibited it at 60 mg/kg, being ineffective in NOP receptor knockout mice. To prove that the bell-shaped profile was mediated by nigral NOP receptors, reverse dialysis of Compound 24 (0.03 μM) in substantia nigra reticulata ameliorated akinesia whereas Compound 24 (3 μM) was ineffective. To demonstrate that motor responses were mediated by tuning inhibitory and excitatory inputs to nigro-thalamic neurons, the low concentration elevated GABA and reduced glutamate in substantia nigra, simultaneously reducing GABA levels in ventro-medial thalamus. Conversely, the higher concentration reduced nigral and elevated thalamic GABA, without affecting nigral glutamate levels. Co-perfusion with raclopride (1 μM) abolished the antiakinetic action of Compound 24 (0.03 μM) and turned the ineffectiveness of Compound 24 (3 μM) into an antiakinetic effect. The low concentration reduced nigral but did not affect thalamic GABA whereas the higher concentration elevated nigral and reduced thalamic GABA. Neither concentration affected nigral glutamate. We conclude that dual motor effects of Compound 24 in hemiparkinsonian rats are accomplished through blockade of nigral NOP receptors resulting in opposite modulation of nigro-thalamic neurons. Endogenous dopamine contributes to these responses affecting the level of GABAergic inhibition of the nigral output via D₂/D₃ receptors.     

Effects of single and multiple treatments with L‐dihydroxyphenylalanine (L‐DOPA) on dopamine receptor–G protein interactions and supersensitive immediate early gene responses in striata of rats after reserpine treatment or with unilateral nigrostriatal lesions

We studied effects of l ‐dihydroxyphenylalanine (L‐DOPA) treatment in rats following reserpine treatment or unilateral 6‐hydroxydopamine (6‐OHDA) injections into medial forebrain bundle. Quantitative in situ hybridization for mRNA's coding for the zinc finger immediate early gene (IEG) zif/268 or Jun family IEG jun b revealed that single L‐DOPA injections accentuated IEG expression 3‐ to 7‐fold in the dopamine (DA)‐depleted striatum. This increased IEG response did not derive from any alterations in DA receptor–G protein coupling, assayed by DA stimulation of ³⁵S‐guanosine‐5′ (γ‐thio) triphosphate (³⁵S‐GTP‐γ‐S) binding to striatal sections. Reserpine treatment increased both basal and maximal striatal DA‐stimulated ³⁵S‐GTP‐γ‐S binding. The augmented IEG responses to single L‐DOPA treatments involved dependency on both D1 and D2 receptors and acutely to N‐methyl‐d ‐aspartate (NMDA) channels. Repetitive L‐DOPA treatments yielded persistently elevated (zif/268) or additionally up‐regulated (jun b) IEG response in the denervated striatum and down‐regulated IEG responses in the control striatum. Degraded L‐DOPA responses and appearance of involuntary movements after chronic L‐DOPA use in advanced Parkinson's disease may derive from these IEG changes. J. Neurosci. Res. 55:71–79, 1999. © 1999 Wiley‐Liss, Inc.