Phosphodiesterase 9 inhibition prolongs the antiparkinsonian action of l-DOPA in parkinsonian non-human primates.

Phosphodiesterase 9 (PDE9) degrades selectively the second messenger cGMP, which is an important molecule of dopamine signaling pathways in striatal projection neurons (SPNs). In this study, we assessed the effects of a selective PDE9 inhibitor (PDE9i) in the primate model of Parkinson's disease (PD). Six macaques with advanced parkinsonism were used in the study. PDE9i was administered as monotherapy and co-administration with l-DOPA at two predetermined doses (suboptimal and threshold s.c. doses of l-Dopa methyl ester plus benserazide) using a controlled blinded protocol to assess motor disability, l-DOPA -induced dyskinesias (LID), and other neurologic drug effects. While PDE9i was ineffective as monotherapy, 2.5 and 5 mg/kg (s.c.) of PDE9i significantly potentiated the antiparkinsonian effects of l-DOPA with a clear prolongation of the "on" state (p < 0.01) induced by either the suboptimal or threshold l-DOPA dose. Co-administration of PDE9i had no interaction with l-DOPA pharmacokinetics. PDE9i did not affect the intensity of LID. These results indicate that cGMP upregulation interacts with dopamine signaling to enhance the l-DOPA reversal of parkinsonian motor disability. Therefore, striatal PDE9 inhibition may be further explored as a strategy to improve motor responses to l-DOPA in PD.

Cortical Serotonergic and Catecholaminergic Denervation in MPTP-Treated Parkinsonian Monkeys.

Decreased cortical serotonergic and catecholaminergic innervation of the frontal cortex has been reported at early stages of Parkinson's disease (PD). However, the limited availability of animal models that exhibit these pathological features has hampered our understanding of the functional significance of these changes during the course of the disease. In the present study, we assessed longitudinal changes in cortical serotonin and catecholamine innervation in motor-symptomatic and asymptomatic monkeys chronically treated with low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Densitometry and unbiased stereological techniques were used to quantify changes in serotonin and tyrosine hydroxylase (TH) immunoreactivity in frontal cortices of 3 control monkeys and 3 groups of MPTP-treated monkeys (motor-asymptomatic [N = 2], mild parkinsonian [N = 3], and moderate parkinsonian [N = 3]). Our findings revealed a significant decrease (P < 0.001) in serotonin innervation of motor (Areas 4 and 6), dorsolateral prefrontal (Areas 9 and 46), and limbic (Areas 24 and 25) cortical areas in motor-asymptomatic MPTP-treated monkeys. Both groups of symptomatic MPTP-treated animals displayed further serotonin denervation in these cortical regions (P < 0.0001). A significant loss of serotonin-positive dorsal raphe neurons was found in the moderate parkinsonian group. On the other hand, the intensity of cortical TH immunostaining was not significantly affected in motor asymptomatic MPTP-treated monkeys, but underwent a significant reduction in the moderate symptomatic group (P < 0.05). Our results indicate that chronic intoxication with MPTP induces early pathology in the corticopetal serotonergic system, which may contribute to early non-motor symptoms in PD.

Microglia, inflammation and gut microbiota responses in a progressive monkey model of Parkinson's disease: A case series.

Inflammation has been linked to the development of nonmotor symptoms in Parkinson's disease (PD), which greatly impact patients' quality of life and can often precede motor symptoms. Suitable animal models are critical for our understanding of the mechanisms underlying disease and the associated prodromal disturbances. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkey model is commonly seen as a "gold standard" model that closely mimics the clinical motor symptoms and the nigrostriatal dopaminergic loss of PD, however MPTP toxicity extends to other nondopaminergic regions. Yet, there are limited reports monitoring the MPTP-induced progressive central and peripheral inflammation as well as other nonmotor symptoms such as gastrointestinal function and microbiota. We report 5 cases of progressive parkinsonism in non-human primates to gain a broader understanding of MPTP-induced central and peripheral inflammatory dysfunction to understand the potential role of inflammation in prodromal/pre-motor features of PD-like degeneration. We measured inflammatory proteins in plasma and CSF and performed [18F]FEPPA PET scans to evaluate translocator proteins (TSPO) or microglial activation. Monkeys were also evaluated for working memory and executive function using various behavior tasks and for gastrointestinal hyperpermeability and microbiota composition. Additionally, monkeys were treated with a novel TNF inhibitor XPro1595 (10 mg/kg, n = 3) or vehicle (n = 2) every three days starting 11 weeks after the initiation of MPTP to determine whether XPro1595 would alter inflammation and microglial behavior in a progressive model of PD. The case studies revealed that earlier and robust [18F]FEPPA PET signals resulted in earlier and more severe parkinsonism, which was seen in male cases compared to female cases. Potential other sex differences were observed in circulating inflammation, microbiota diversity and their metabolites. Additional studies with larger group sizes of both sexes would enable confirmation and extension of these findings. If these findings reflect potential differences in humans, these sex differences have significant implications for therapeutic development of inflammatory targets in the clinic.

Group I metabotropic glutamate receptors in the primate motor thalamus: subsynaptic association with cortical and sub-cortical glutamatergic afferents.

Preclinical evidence indicates that mGluR5 is a potential therapeutic target for Parkinson's disease and L-DOPA-induced dyskinesia. However, the mechanisms through which these therapeutic benefits are mediated remain poorly understood. Although the regulatory role of mGluR5 on glutamatergic transmission has been examined in various basal ganglia nuclei, very little is known about the localization and function of mGluR5 in the ventral motor and intralaminar thalamic nuclei, the main targets of basal ganglia output in mammals. Thus, we used immuno-electron microscopy to map the cellular and subcellular localization of group I mGluRs (mGluR1a and mGluR5) in the ventral motor and caudal intralaminar thalamic nuclei in rhesus monkeys. Furthermore, using double immuno-electron microscopy, we examined the subsynaptic localization of mGluR5 in relation to cortical and sub-cortical glutamatergic afferents. Four major conclusions can be drawn from these data. First, mGluR1a and mGluR5 are expressed postsynaptically on the plasma membrane of dendrites of projection neurons and GABAergic interneurons in the basal ganglia- and cerebellar-receiving regions of the ventral motor thalamus and in CM. Second, the plasma membrane-bound mGluR5 immunoreactivity is preferentially expressed perisynaptically at the edges of cortical and sub-cortical glutamatergic afferents. Third, the mGluR5 immunoreactivity is more strongly expressed in the lateral than the medial tiers of CM, suggesting a preferential association with thalamocortical over thalamostriatal neurons in the primate CM. Overall, mGluR5 is located to subserve powerful modulatory role of cortical and subcortical glutamatergic transmission in the primate ventral motor thalamus and CM.

Metabotropic glutamate receptors: targets for neuroprotective therapies in Parkinson disease.

Metabotropic glutamate receptors (mGluRs) are heavily expressed throughout the basal ganglia (BG), where they modulate neuronal excitability, transmitter release and long term synaptic plasticity. Therefore, targeting specific mGluR subtypes by means of selective drugs could be a possible strategy for restoring normal synaptic function and neuronal activity of the BG in Parkinson disease (PD). Preclinical studies have revealed that specific mGluR subtypes mediate significant neuroprotective effects that reduce toxin-induced midbrain dopaminergic neuronal death in animal models of PD. Although the underlying mechanisms of these effects must be further studied, there is evidence that intracellular calcium regulation, anti-inflammatory effects, and glutamatergic network modulation contribute to some of these neuroprotective properties. It is noteworthy that these protective effects extend beyond midbrain dopaminergic neurons to include other monoaminergic cell groups for some mGluRs. In this review, we discuss evidence for mGluR-mediated neuroprotection in PD and highlight the challenges to translate these findings into human trials.

Chronic MPTP administration regimen in monkeys: a model of dopaminergic and non-dopaminergic cell loss in Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder clinically characterized by cardinal motor deficits including bradykinesia, tremor, rigidity and postural instability. Over the past decades, it has become clear that PD symptoms extend far beyond motor signs to include cognitive, autonomic and psychiatric impairments, most likely resulting from cortical and subcortical lesions of non-dopaminergic systems. In addition to nigrostriatal dopaminergic degeneration, pathological examination of PD brains, indeed, reveals widespread distribution of intracytoplasmic inclusions (Lewy bodies) and death of non-dopaminergic neurons in the brainstem and thalamus. For that past three decades, the MPTP-treated monkey has been recognized as the gold standard PD model because it displays some of the key behavioral and pathophysiological changes seen in PD patients. However, a common criticism raised by some authors about this model, and other neurotoxin-based models of PD, is the lack of neuronal loss beyond the nigrostriatal dopaminergic system. In this review, we argue that this assumption is largely incorrect and solely based on data from monkeys intoxicated with acute administration of MPTP. Work achieved in our laboratory and others strongly suggest that long-term chronic administration of MPTP leads to brain pathology beyond the dopaminergic system that displays close similarities to that seen in PD patients. This review critically examines these data and suggests that the chronically MPTP-treated nonhuman primate model may be suitable to study the pathophysiology and therapeutics of some non-motor features of PD.

Reduced noradrenergic innervation of ventral midbrain dopaminergic cell groups and the subthalamic nucleus in MPTP-treated parkinsonian monkeys.

There is anatomical and functional evidence that ventral midbrain dopaminergic (DA) cell groups and the subthalamic nucleus (STN) receive noradrenergic innervation in rodents, but much less is known about these interactions in primates. Degeneration of NE neurons in the locus coeruleus (LC) and related brainstem NE cell groups is a well-established pathological feature of Parkinson's disease (PD), but the development of such pathology in animal models of PD has been inconsistent across species and laboratories. We recently demonstrated 30-40% neuronal loss in the LC, A5 and A6 NE cell groups of rhesus monkeys rendered parkinsonian by chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study, we used dopamine-beta-hydroxylase (DßH) immunocytochemistry to assess the impact of this neuronal loss on the number of NE terminal-like varicosities in the substantia nigra pars compacta (SNC), ventral tegmental area (VTA), retrorubral field (RRF) and STN of MPTP-treated parkinsonian monkeys. Our findings reveal that the NE innervation of the ventral midbrain and STN of normal monkeys is heterogeneously distributed being far more extensive in the VTA, RRF and dorsal tier of the SNC than in the ventral SNC and STN. In parkinsonian monkeys, all regions underwent a significant (~50-70%) decrease in NE innervation. At the electron microscopic level, some DßH-positive terminals formed asymmetric axo-dendritic synapses in VTA and STN. These findings demonstrate that the VTA, RRF and SNCd are the main ventral midbrain targets of ascending NE inputs, and that these connections undergo a major break-down in chronically MPTP-treated parkinsonian monkeys. This severe degeneration of the ascending NE system may contribute to the pathophysiology of ventral midbrain and STN neurons in PD.

Lack of Antiparkinsonian Effects of Systemic Injections of the Specific T-Type Calcium Channel Blocker ML218 in MPTP-Treated Monkeys.

Dopaminergic medications ameliorate many of the motor impairments of Parkinson's disease (PD). However, parkinsonism is often only partially reversed by these drugs, and they can have significant side effects. Therefore, a need remains for novel treatments of parkinsonism. Studies in rodents and preliminary clinical evidence have shown that T-type calcium channel (TTCC) antagonists have antiparkinsonian effects. However, most of the available studies utilized nonselective agents. We now evaluated whether systemic injections of the specific TTCC blocker ML218 have antiparkinsonian effects in MPTP-treated parkinsonian Rhesus monkeys. The animals were treated chronically with MPTP until they reached stable parkinsonism. In pharmacokinetic studies, we found that ML218 reaches a peak CSF concentration 1-2 h after s.c. administration. In electrocardiographic studies, we found no effects of ML218 on cardiac rhythmicity. As expected, systemic injections of the dopamine precursor L-DOPA dose-dependently increased the movements in our parkinsonian animals. We then tested the behavioral effects of systemic injections of ML218 (1, 10, or 30 mg/kg) or its vehicle, but did not detect specific antiparkinsonian effects. ML218 (3 or 10 mg/kg) was also not synergistic with L-DOPA. Using recordings of electrocorticogram signals (in one animal), we found that ML218 increased sleep. We conclude that ML218 does not have antiparkinsonian effects in MPTP-treated parkinsonian monkeys, due at least in part, to the agent's sedative effects.

Effects of a novel phosphodiesterase 10A inhibitor in non-human primates: A therapeutic approach for schizophrenia with improved side effect profile.

Schizophrenia symptoms are associated with alterations in basal ganglia-cortical networks that include the cyclic nucleotides (cAMP/cGMP) signaling pathways. Phosphodiesterase 10A (PDE10A) inhibitors have been considered as therapeutic agents for schizophrenia because the regulation of cAMP and cGMP in the striatum by PDE10A plays an important role in the signaling mechanisms of the striatal-cortical network, and thereby in cognitive function. In the present study we assessed in non-human primates (NHPs) the effects of a novel PDE10A inhibitor (FRM-6308) that has demonstrated high potency and selectivity for human recombinant PDE10A in vitro. The behavioral effects of FRM-6308 in a dose range were determined in rhesus monkeys using a standardized motor disability scale for primates, motor tasks, and the "drug effects on the nervous system" (DENS) scale. The neuronal metabolic effects of FRM-6308 were determined with [(18)F]-fluorodeoxyglucose PET imaging. Results showed that FRM-6308 did not have any specific effects on the motor system at s.c. doses up to 0.32 mg/kg in NHPs, which induced a significant increase in the FDG-SUV in striatum (F 16.069, p < 0.05) and cortical (F 15.181, p < 0.05) regions. Higher doses induced sedation and occasional involuntary movements with clear development of tolerance after repeated exposures. These findings suggest that FRM-6308 has the adequate pharmacological profile to advance testing in clinical trials and demonstrate antipsychotic efficacy of PDE10A inhibition for the treatment of schizophrenia patients.

Phosphodiesterase 10A inhibitor MP-10 effects in primates: comparison with risperidone and mechanistic implications.

Phosphodiesterase 10A (PDE10A) is highly expressed in striatal medium spiny neurons of both the direct and indirect output pathways. Similar to dopamine D2 receptor antagonists acting on indirect pathway neurons, PDE10A inhibitors have shown behavioral effects in rodent models that predict antipsychotic efficacy. These findings have supported the clinical investigation of PDE10A inhibitors as a new treatment for schizophrenia. However, PDE10A inhibitors and D2 antagonists differ in effects on direct pathway and other neurons of the basal ganglia, indicating that these two drug classes may have divergent antipsychotic efficacy and side effect profile. In the present study, we compare the behavioral effects of the selective PDE10A inhibitor MP-10 to those of the clinical standard D2 antagonist risperidone in rhesus monkeys using a standardized motor disability scale for parkinsonian primates and a newly designed "Drug Effects on Nervous System" scale to assess non-motor effects. Behavioral effects of MP-10 correlated with its plasma levels and its regulation of metabolic activity in striatal and cortical regions as measured by FDG-PET imaging. While MP-10 and risperidone broadly impacted similar behavioral domains in the primate, their effects had a different underlying basis. MP-10-treated animals retained the ability to respond but did not engage tasks, whereas risperidone-treated animals retained the motivation to respond but were unable to perform the intended actions. These findings are discussed in light of what is currently known about the modulation of striatal circuitry by these two classes of compounds, and provide insight into interpreting emerging clinical data with PDE10A inhibitors for the treatment of psychotic symptoms.

Extrastriatal D2-like receptors modulate basal ganglia pathways in normal and Parkinsonian monkeys.

According to traditional models of the basal ganglia-thalamocortical network of connections, dopamine exerts D2-like receptor (D2LR)-mediated effects through actions on striatal neurons that give rise to the "indirect" pathway, secondarily affecting the activity in the internal and external pallidal segments (GPi and GPe, respectively) and the substantia nigra pars reticulata (SNr). However, accumulating evidence from the rodent literature suggests that D2LR activation also directly influences synaptic transmission in these nuclei. To further examine this issue in primates, we combined in vivo electrophysiological recordings and local intracerebral microinjections of drugs with electron microscopic immunocytochemistry to study D2LR-mediated modulation of neuronal activities in GPe, GPi, and SNr of normal and MPTP-treated (parkinsonian) monkeys. D2LR activation with quinpirole increased firing in most GPe neurons, likely due to a reduction of striatopallidal GABAergic inputs. In contrast, local application of quinpirole reduced firing in GPi and SNr, possibly through D2LR-mediated effects on glutamatergic inputs. Injections of the D2LR antagonist sulpiride resulted in effects opposite to those of quinpirole in GPe and GPi. D2 receptor immunoreactivity was most prevalent in putative striatal-like GABAergic terminals and unmyelinated axons in GPe, GPi, and SNr, but a significant proportion of immunoreactive boutons also displayed ultrastructural features of glutamatergic terminals. Postsynaptic labeling was minimal in all nuclei. The D2LR-mediated effects and pattern of distribution of D2 receptor immunoreactivity were maintained in the parkinsonian state. Thus, in addition to their preferential effects on indirect pathway striatal neurons, extrastriatal D2LR activation in GPi and SNr also influences direct pathway elements in the primate basal ganglia under normal and parkinsonian conditions.

Sequestration of toxic oligomers by HspB1 as a cytoprotective mechanism.

Small heat shock proteins (sHsps) are molecular chaperones that protect cells from cytotoxic effects of protein misfolding and aggregation. HspB1, an sHsp commonly associated with senile plaques in Alzheimer's disease (AD), prevents the toxic effects of Aß aggregates in vitro. However, the mechanism of this chaperone activity is poorly understood. Here, we observed that in two distinct transgenic mouse models of AD, mouse HspB1 (Hsp25) localized to the penumbral areas of plaques. We have demonstrated that substoichiometric amounts of human HspB1 (Hsp27) abolish the toxicity of Aß oligomers on N2a (mouse neuroblastoma) cells. Using biochemical methods, spectroscopy, light scattering, and microscopy methods, we found that HspB1 sequesters toxic Aß oligomers and converts them into large nontoxic aggregates. HspB1 was overexpressed in N2a cells in response to treatment with Aß oligomers. Cultured neurons from HspB1-deficient mice were more sensitive to oligomer-mediated toxicity than were those from wild-type mice. Our results suggest that sequestration of oligomers by HspB1 constitutes a novel cytoprotective mechanism of proteostasis. Whether chaperone-mediated cytoprotective sequestration of toxic aggregates may bear clues to plaque deposition and may have potential therapeutic implications must be investigated in the future.

Metabotropic glutamate receptor 5 antagonist protects dopaminergic and noradrenergic neurons from degeneration in MPTP-treated monkeys.

Degeneration of the dopaminergic nigrostriatal system and of noradrenergic neurons in the locus coeruleus are important pathological features of Parkinson's disease. There is an urgent need to develop therapies that slow down the progression of neurodegeneration in Parkinson's disease. In the present study, we tested whether the highly specific metabotropic glutamate receptor 5 antagonist, 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine, reduces dopaminergic and noradrenergic neuronal loss in monkeys rendered parkinsonian by chronic treatment with low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Weekly intramuscular 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injections (0.2-0.5 mg/kg body weight), in combination with daily administration of 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine or vehicle, were performed until the development of parkinsonian motor symptoms in either of the two experimental groups (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine versus 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/vehicle). After 21 weeks of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment, all 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/vehicle-treated animals displayed parkinsonian symptoms, whereas none of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine-treated monkeys were significantly affected. These behavioural observations were consistent with in vivo positron emission tomography dopamine transporter imaging data, and with post-mortem stereological counts of midbrain dopaminergic neurons, as well as striatal intensity measurements of dopamine transporter and tyrosine hydroxylase immunoreactivity, which were all significantly higher in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine-treated animals than in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/vehicle-treated monkeys. The 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine treatment also had a significant effect on the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced loss of norepinephrine neurons in the locus coeruleus and adjoining A5 and A7 noradrenaline cell groups. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/vehicle-treated animals, almost 40% loss of tyrosine hydroxylase-positive norepinephrine neurons was found in locus coeruleus/A5/A7 noradrenaline cell groups, whereas the extent of neuronal loss was lower than 15% of control values in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine-treated monkeys. Our data demonstrate that chronic treatment with the metabotropic glutamate receptor 5 antagonist, 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine, significantly reduces 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity towards dopaminergic and noradrenergic cell groups in non-human primates. This suggests that the use of metabotropic glutamate receptor 5 antagonists may be a useful strategy to reduce degeneration of catecholaminergic neurons in Parkinson's disease.

(18)F-FECNT: validation as PET dopamine transporter ligand in parkinsonism.

The positron emission tomography (PET) tracer 2ß-carbomethoxy-3ß-(4-chlorophenyl)-8-(2-[18F]-fluoroethyl)-nortropane ((18)F-FECNT) is a highly specific ligand for dopamine transporter (DAT) that yields higher peak striatum-to-cerebellum ratios and offers more favorable kinetics than most (18)F-radiolabeled DAT ligands currently available. The goal of this study is to validate the use of (18)F-FECNT as a PET radiotracer to assess the degree of striatal dopamine terminals denervation and midbrain dopaminergic cell loss in MPTP-treated parkinsonian monkeys. Three rhesus monkeys received weekly injections of MPTP (0.2-0.5 mg/kg) for 21 weeks, which resulted in the progressive development of a moderate level of parkinsonism. We carried out (18)F-FECNT PET at baseline (twice; 10 weeks apart) and at week 21 post-MPTP injections. Postmortem stereological cell counts of dopaminergic neurons in the ventral midbrain, and intensity measurements of DAT and tyrosine hydroxylase (TH) immunoreactivity in the striatum were performed and correlated with striatal and ventral midbrain PET data. Three additional monkeys were used as controls for midbrain dopaminergic cell counts, and striatal DAT or TH immunoreactivity measurements. The correlation and coefficient of variance between (18)F-FECNT test-retest specific uptake ratios were 0.99 (R²) and 2.65%, respectively. The (18)F-FECNT binding potential of the ventral midbrain and striatal regions was tightly correlated with postmortem stereological cell counts of nigral dopaminergic neurons (R²=0.91), and striatal DAT (R²=0.83) or TH (R²=0.88) immunoreactivity intensity measurements. These findings demonstrate that (18)F-FECNT is a highly sensitive PET imaging ligand to quantify both striatal dopamine denervation and midbrain dopaminergic cell loss associated with parkinsonism.

Lethal and sub-lethal effects of chlorination on green mussel Perna viridis in the context of biofouling control in a power plant cooling water system.

Continuous chlorination is a widely followed cooling water treatment practice used in the power industry to combat biofouling. The green mussel Perna viridis is one of the dominant fouling organisms ( > 70%) in the Madras Atomic Power Station. Mortality pattern as well as physiological responses such as oxygen consumption, filtration rate, byssus thread production and faecal matter production of three different size groups of this mussel were studied at different chlorination concentrations. At 0.7 mg l(-1) residual chlorine, 3-4 cm size mussels showed 100% mortality in 553.3 h while 8-9 cm size group mussels died within 588 h. At a relatively high level of residual chlorine (9.1 mg l(-1)), 100% mortality in 3-4 cm and 8-9 cm size groups took 94 and 114 h, respectively. All physiological activities studied showed a progressive reduction as chlorine residuals were increased from 0 to 0.55 mg l(-1). The data indicated that the green mussel can sense a residual chlorine level as low as < 0.15 mg l(-1) and complete valve closure occurs only at 0.55 mg l(-1). The paper also shows that the sub-lethal physiological responses are better indices than lethal responses in planning chlorination strategies.