Selective serotonin reuptake inhibitors (SSRIs) represent a class of pharmaceuticals previously reported in aquatic ecosystems. SSRIs are designed to treat depression and other disorders in humans, but are recognized to elicit a variety of effects on aquatic organisms, ranging from neuroendocrine disruption to behavioral perturbations. However, an understanding of the relationships among mechanistic responses associated with SSRI targets and ecologically important behavioral responses of fish remains elusive. Herein, linking Adverse Outcomes Pathways (AOP) models with internal dosimetry represent potential approaches for developing an understanding of pharmaceutical risks to aquatic life. We selected sertraline as a model SSRI for a 28-d study with adult male fathead minnows. Binding activity of the serotonin reuptake transporter (SERT), previously demonstrated in mammals and fish models to respond to sertraline exposure, was selected as an endpoint associated with therapeutic activity. Shelter-seeking behavior was monitored using digital tracking software to diagnose behavioral abnormalities. Fish plasma levels of sertraline exceeding human therapeutic doses were accurately modeled from external exposure concentrations when pH influences on ionization and log D were considered. We observed statistically significant decreases in binding at the therapeutic target (SERT) and shelter-seeking behavior when fish plasma levels exceeded human therapeutic thresholds. Such observations highlights the strengths of coupling physiologically based pharmacokinetic modeling and AOP approaches and suggest that internal dosimetry should be monitored to advance an understanding of the ecological consequences of SSRI exposure to aquatic vertebrates.

No abstract

Modulation of excitatory synaptic transmission by presynaptic metabotropic glutamate receptors (mGluRs) was examined in brain slices from control rats and rats with amygdala-kindled seizures. Using whole-cell voltage-clamp and current-clamp recordings, this study shows for the first time that in control and kindled basolateral amygdala neurons, two pharmacologically distinct presynaptic mGluRs mediate depression of synaptic transmission. Moreover, in kindled neurons, agonists at either group II- or group III-like mGluRs exhibit a 28- to 30-fold increase in potency and suppress synaptically evoked bursting. The group II mGluR agonist (2S,3S,4S)-2-(carboxycyclopropyl)glycine (L-CCG) dose-dependently depressed monosynaptic EPSCs evoked by stimulation in the lateral amygdala with EC50 values of 36 nM (control) and 1.2 nM (kindled neurons). The group III mGluR agonist L-2-amino-4-phosphonobutyrate (L-AP4) was less potent, with EC50 values of 297 nM (control) and 10.8 nM (kindled neurons). The effects of L-CCG and L-AP4 were fully reversible. Neither L-CCG (0.0001-10 microM) nor L-AP4 (0.001-50 microM) caused membrane currents or changes in the current-voltage relationship. The novel mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)-glycine (MCCG; 100 microM) and (S)-2-methyl-2-amino-4-phosphonobutyrate (MAP4; 100 microM) selectively reversed the inhibition by L-CCG and L-AP4 to 81.3 +/- 12% and 65.3 +/- 6.6% of predrug, respectively. MCCG and MAP4 (100-300 microM) themselves did not significantly affect synaptic transmission. The exquisite sensitivity of agonists in the kindling model of epilepsy and the lack of evidence for endogenous receptor activation suggest that presynaptic group II- and group III-like mGluRs might be useful targets for suppression of excessive synaptic activation in neurological disorders such as epilepsy.

1. Inward currents evoked by metabotropic glutamate receptor (mGlu) agonists quisqualate and 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) were characterized in the basolateral nucleus of the amygdala. Currents were recorded with whole-cell patch electrodes in the presence of D-2-amino-5-phosphonovaleric acid (D-APV, 50 uM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 30 uM) and tetrodotoxin (TTX, 1 uM). 2. When recording with K+ electrodes, quisqualate (10-50 uM) produced an inward current which was not associated with a significant change in membrane slope conductance (Gm) and was insensitive to Ba2+ (0-2 mM) and Cs+ (2 mM). The 1S,3R-ACPD (50-200 /uM)-induced inward current was associated with a decreased 0m and reversed polarity around -95 mV.However, in BaP+ and Cs+, the 1S,3R-ACPD inward current amplitude was enhanced and was not accompanied by a change in Gm, a response similar to that evoked by quisqualate. 3. Glutamate (1 mM) and the group I mGlu specific agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 100 sM) also evoked currents not associated with a change in Gm. 4. When recorded with Cs+ electrodes in external Ba2+ and Cs+ solution, quisqualate activated an inward current more potently than 1S,3R-ACPD, suggesting that this current is preferentially activated by quisqualate. The mGlu agonist-induced inward current was not accompanied by a Gm change under these conditions. 5. Substitution of extracellular Nae with Li+ (117 or 50 mM) or with 100 mm choline reduced the quisqualate-and IS,3R-ACPD-induced inward currents, results consistent with mediation by Na+-Ca2+ exchange. 6. The quisqualate-and IS,3R-ACPD-induced inward currents were reduced in Ca2+-free EGTA (1 mM) solution and prevented by including the Ca2P chelating agent BAPTA (10 mM) in the recording electrode. In low-Ca2+ (100 /M)-and Cd2+ (200 uM)-containing solution to block voltage-gated Ca2P currents, the quisqualate-induced current was not altered, but the 1S,3R-ACPD inward current was blocked. These data suggest that the quisqualate-and 1S,3R-ACPD-induced currents are mediated through a rise in intracellular Ca2+ and require extracellular Ca2+ but that the 1S,3R-ACPD current may depend on Ca2P influx via voltagegated Ca2P channels. 7. The quisqualate current with no Gm change was inhibited by including the Nae-Ca2P exchange inhibitory peptide (XIP; 10/uM) in the K+ recording electrode. XIP did not prevent the outward current evoked by baclofen (10 uM) or the 1S,3R-ACPD-induced inward current associated with decreased conductance.8. These data are consistent with the hypothesis that quisqualate and 1S,3R-ACPD in Ba2P and Cs+ solution activate a Na+-Ca2+ exchange current not associated with a conductance change. The quisqualate exchange current mediated through a group I mGlu may result from mobilization of Ca2+ from intracellular stores. The 1S,3R-ACPD exchange current requires extracellular Ca2+ passing through voltage-gated Ca2+ channels and may be mediated through a different receptor.

1. Metabotropic glutamate receptor (mGluR)-agonist-induced hyperpolarizations and corresponding outward currents were analyzed in basolateral amygdala (BLA) neurons in rat brain slice preparations with current-clamp and single-electrode voltage-clamp recording to characterize the mGluR subtype(s) and the ion channel(s) mediating this response. 2. The mGluR agonist (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) induced a membrane hyperpolarization or outward current in BLA neurons in a concentration-dependent manner (median effective concentration = 34 microM; range = 10-200 microM); the 1S,3R-ACPD hyperpolarizations are recorded in 89% of neurons that accommodate or cease firing in response to a 400-ms depolarizing current injection (0.5 nA). 3. mGluR agonists elicited hyperpolarizations or outward currents in a concentration-dependent manner in the following rank order of potency: (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I) > 1S,3R-ACPD > (s)-4-carboxyphenylglycine = (RS)-4-carboxy-3-hydroxyphenylglycine (4C3HPG) > L-aminophosphonobutyric acid > (1S,3S)-1-amino-cyclopentane-1,3-dicarboxylic acid. In contrast, the mGluR agonists quisqualate and ibotenate induced only depolarizations in the presence of D-2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2,3-dione in BLA neurons. 4. The 1S,3R-ACPD-induced outward current is mediated through a large-conductance calcium-dependent potassium (BK) conductance. The BK channel blockers iberiotoxin and charybdotoxin blocked the response, as did the potassium channel blockers tetraethylammonium and 4-aminopyridine; the small-conductance calcium-activated potassium channel blocker apamin did not affect the response. 5. The mGluR-agonist-induced hyperpolarization is blocked in amygdala slices from animals pretreated with pertussis toxin (PTX). 1S,3R-ACPD hyperpolarizations were recorded in neurons contralateral but not ipsilateral to the site of PTX injection. 6. The antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG, 500 microM) reduced significantly the 1S,3R-ACPD-induced hyperpolarization. 7. In conclusion, the relative potency of L-CCG-I and 4C3HPG in evoking only hyperpolarizations (outward currents) in accommodating neurons, and the observation that MCPG (500 microM) reduces the hyperpolarization, suggest that a group-II-like mGluR underlies the hyperpolarizing response. The mGluR-induced response is sensitive to iberiotoxin and to pretreatment with PTX, suggesting activation of BK channels through a group II mGluR linked to a PTX-sensitive G protein in BLA neurons.