Characterization of strychnine-sensitive glycine receptors in acutely isolated adult rat basolateral amygdala neurons

Brain Research 859 Ž2000. 341–351 www.elsevier.comrlocaterbres

Research report

Characterization of strychnine-sensitive glycine receptors in acutely isolated adult rat basolateral amygdala neurons Brian A. McCool ) , Shaleen K. Botting Department of Medical Pharmacology and Toxicology, Texas A & M UniÕersity System Health Science Center, 368 Reynolds Building, College Station, TX 77843, USA Accepted 11 January 2000

Abstract Large concentrations of the b-amino acid, taurine, can be found in many forebrain areas such as the basolateral amygdala, a portion of the limbic forebrain intimately associated with the regulation of fearranxiety-like behaviors. In addition to its cytoprotective and osmoregulatory roles, taurine may also serve as an agonist at GABA A- and strychnine-sensitive glycine receptors. In this latter context, the present study demonstrates that application of taurine to acutely isolated neurons from the basolateral amygdala of adult rats causes significant alterations in resting membrane current, as measured by whole-cell patch clamp electrophysiology. Using standard pharmacological approaches, we find that currents gated by concentrations of taurine F 3 mM are predominantly mediated by strychnine-sensitive receptors. Furthermore, these strychnine-sensitive receptors are shown to be pharmacologically and biophysically similar to ‘classic’ strychnine-sensitive, chloride-conducting glycine receptors expressed in brainstem and spinal cord. While amygdala glycine receptors can be distinguished from GABA A receptors expressed by the same neurons, these two chloride channels are functionally expressed at comparable levels. Given that a number of clinically relevant compounds are associated with the regulation of GABA A receptors in this brain region, the presence of both strychnine-sensitive glycine receptors and their agonist, taurine, in the basolateral amygdala may suggest an important role for these receptors in the limbic forebrain of adult rats. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Electrophysiology; Basolateral amygdala; Strychnine; Glycine receptor

1. Introduction As part of the limbic system, the amygdala plays a highly integrative role in behavioral regulation. Its afferent and efferent systems connect cognitive, sensory, and ‘central autonomic control’ brain regions, thus occupying a pivotal position in the sensermemory–integration–response pathway. In humans and non-human primates Žreviewed in Refs. w13,45x., the amygdala appears to regulate behaviors like social interaction, aggression, and fearr anxiety. Such findings have also been extended to more experimental models like the rat where many of the behavioral responses to fearrapprehension-inducing stimuli are very similar to those in humans w8x. Of particular relevance for the studies outlined below, rat models of fearranxiety have implicated one particular amygdala subdivision, the basolateral nucleus ŽBLA., as being centrally important in ) Corresponding author. [email protected]

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q1-409-845-0699;

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both the acquisition and expression of fearrapprehensionrelated behaviors Žreviewed in Ref. w9x.. Because of this central, integrative niche played by the BLA, an intimate understanding of the neurotransmitter systems governing neuronal excitability will provide important insight into the neurophysiological basis of many sensory-related, amygdala-dependent behaviors, particularly fear and anxiety. While GABA A receptor activation in the BLA has been shown to mediate the anxiolytic activity of benzodiazepines w12,38,39x, relatively little is known about other neurotransmitter systems involved in the regulation of fearranxiety-related behaviors Žbut see Refs. w12,23,37x.. Recently, it has been demonstrated in rats that acute administration of ethanol, an intoxicant whose anxiolytic properties are well-established w3x, can cause significant increases in extracellular concentrations of taurine in the basolateral amygdala w31,32x. The physiologic role of taurine is multifaceted Žreviewed in Ref. w17x., mirrored by the multitude of conditions, such as experimentally induced status epilepticus w48x, iso-osmotic elevated extracel-

0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 0 0 . 0 2 0 2 6 - 6

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lular Kq w10,27x, hypoglycemia w43x, ischemia w2x, and direct ionotropic glutamate receptor activation w27x, that are known to instigate its release from intracellular storage sites. While such findings are often interpreted in the context of taurine’s activity as a cellular osmolyte w17x, this b-amino acid is also an agonist for neurotransmitter receptors, namely glycine- and GABA-gated chloride channels w15,50x. Importantly, the neurons of the basolateral amygdala not only express GABA A receptors w25x but appear to concentrate taurine w28x, suggesting that taurine may play a neurotransmitter role in this brain area. However, the neuromodulatory potential of taurine release in the basolateral amygdala has not been directly examined. As an initial step towards understanding any neurotransmitter role for taurine in the basolateral amygdala, we have used whole-cell patch-clamp electrophysiology to investigate the effects of this amino acid on resting membrane properties in acutely isolated basolateral amygdala neurons from adult rats. We find that taurine not only interacts with GABA A receptors but also defines a neurotransmitter receptor not yet described in this limbic forebrain area. Our investigations into the characteristics of this receptor provide some insights into the neurophysiology of both taurine- and chloride-conducting neurotransmitter receptors in the basolateral amygdala.

mM.: 130 N-methyl glucamine, 10 NaCl, 1 MgCl 2 , 10 HEPES, 10 D-glucose; pH 7.4 with HCl, osmolality 325 mmolrkg adjusted with sucrose. and those regions containing primarily basolateral amygdala Žsee Fig. 1A. were carefully dissected away from the remaining tissues. Individual neurons were isolated from these tissue pieces by mechanical separation using fire-polished Pasteur pipettes. The dispersed tissue was transferred to plastic coverslips ŽThemonox.. Large neurons Ž15–35 pF. with pyramidal or stellate soma ŽFig. 1B1 and B 2 . were utilized exclusively in these studies and had morphological characteristics that

2. Materials and methods 2.1. Preparation of coronal brain slices Adult male rats Ž198 " 13 g, n s 45. were anesthetized with isoflurane and decapitated in accordance with the NIH Guide for the Care and Use of Laboratory Animals using a protocol approved by the Texas A & M University Laboratory Animal Care Committee. The brain was rapidly removed and chilled with oxygenated, ice-cold ‘high Mg 2qrlow Ca2q artificial cerebrospinal fluid ŽaCSF.’ containing Žin mM.: 125 NaCl, 5 KCl, 25 NaHCO 3 , 1.25 NaH 2 PO4 , 1 MgSO4 , 1.5 MgCl 2 , 0.5 CaCl 2 , and 20 D-glucose. Coronal sections containing the lateralrbasolateral amygdala were made with a manual Vibroslice ŽCambden Inst.. and were held in this modified aCSF bubbled with 95% O 2r5% CO 2 at room temperature until use Žtypically 1–6 h.. 2.2. Isolation of neurons from brain slices Neurons were isolated from coronal brain slices containing the lateralrbasolateral amygdala according to published procedures w4x. Briefly, slices were digested with 0.5–1 mgrml Pronase ŽCalbiochem. dissolved in standard aCSF Žsimilar to the ‘modified’ aCSF, except containing 1 mM MgSO4 , 2 mM CaCl 2 , without MgCl 2 . at 378C for 20 min with constant oxygenation. Following this digestion, slices were removed to ‘isolation buffer’ Žcontaining Žin

Fig. 1. Isolation of basolateral amygdala neurons. ŽA. Following exposure to protease, the basolateral amygdala was dissected from coronal brain slices using a scalpel. Lines indicate approximate cut sites. Level: 2.8 mm posterior of the Bregma. Adapted from Paxinos and Watson w29x. Abbreviations: Ce — central amygdaloid nucleus; La — lateral amygdaloid nucleus; Bla — basolateral amygdaloid nucleus Žanterior and posterior components.; Blv — ventral basolateral nucleus; Bma — basomedial amygdaloid nucleus Žanterior.; En — dorsal endopiriform nucleus. ŽB1 and B 2 . Typical morphology of acutely isolated basolateral amygdala neurons consisted of pyramidal ŽB1 . or fusiform ŽB 2 . soma and three to five apical dendrites. The average size of the neuronal soma can be approximated by the mean membrane capacitance which was 23.7"1.1 pF Žaverage"S.E.M., ns 26. for the neurons in this study where the membrane capacitance was measured by a computer algorithm Žsee Section 2..

B.A. McCool, S.K. Bottingr Brain Research 859 (2000) 341–351

were similar to both isolated BLA neurons w46x and BLA neurons in situ w22x.

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tionships; and Ymin s 0 for the taurine and b-alanine concentration–response relationships.

2.3. Whole-cell electrophysiology 3. Results The whole-cell patch-clamp technique was performed on acutely dissociated neurons that were continuously perfused with a HEPES-buffered saline ŽHBS. solution Žcontaining Žin mM.: 150 NaCl, 10 HEPES, 2.5 KCl, 2.5 CaCl 2 , 1.0 MgCl 2 , 10 D-glucose, 0.0005 TTX; pH 7.4 with NaOH, osmolality 320 mmolrkg adjusted with sucrose.. Drugs were diluted from concentrated stocks into HBS and applied within 100 mm of the cell using a linear array of fused silica tubes Ž150 mm i.d.; Hewlett Packard. mounted on a manipulator. A Csq-based internal solution Žin mM: 130 CsCl, 10 HEPES, 10 EGTA, 1 CaCl 2 , 4 Mg-ATP; pH 7.2 with CsOH, osmolality 305 mmolrkg adjusted with sucrose. was used. In some experiments, this internal solution was modified so that the 130 mM CsCl was replaced with 110 mM CsMeSO4 and 20 mM CsCl. Recordings were done at room temperature according to published procedures w14,21x using an Axopatch ID amplifier ŽAxon Instruments. in voltage-clamp mode. In some neurons, whole-cell capacitance Ž24.9 " 0.9 pF, n s 48. and series resistance Ž21.2 " 1.4 M V, n s 48. were determined by fits of the capacitive transients during squarewave voltage steps using standard software algorithms contained within pClamp 7.0 software ŽAxon Instruments.. In every neuron, series resistance was monitored throughout the recording but was not compensated. Data were passed through a three-pole low-pass Bessel filter Ž0.5 kHz., digitized at 1 kHz ŽDigiData 1200, Axon Instruments., and stored on a microcomputer until analyzed. Data analysis was performed off-line using Clampfit ŽAxon Instruments. and Prism ŽGraphPad. software. Where indicated, current amplitudes are reported as values standardized to the whole-cell capacitance Žin pF.. Summarized data in the text are reported as the mean " S.E.M. For concentration–response relationships, peak current responses at different concentrations of agonists or antagonist were normalized to a response from a maximally efficacious Ž1 mM, agonists. or ; EC 50 Ž50 mM, antagonist. concentration of glycine. Normalized data were plotted vs. the logarithm of the concentration of agonistrantagonist. To determine the EC 50 or IC 50 and Hill slope Ž n H ., plots were fit ŽPrism v. 2.01, GraphPad Software. to the ‘four-parameter logistic equation’: Y s Ymin q

Ymax y Ymin 1 q 10 Žlog EC 50 yX . n H

,

where Y s response expressed as percent control; X s logŽwagonistrantagonistx., and Hill slope Ž n H . s slope of the concentration–response relationship. Because data in each neuron were normalized, Ymin s 0 and Ymax s 100 for the glycine and strychnine concentration–response rela-

3.1. Taurine-gated currents in acutely isolated basolateral amygdala neurons To determine whether the b-amino acid, taurine, could act as a neurotransmitter receptor agonist, relatively high concentrations Ž3 and 10 mM. were applied to acutely isolated basolateral amygdala neurons during whole-cell recordings. From a holding potential of y60 mV and using high intracellular Cly concentrations, taurine elicited inward currents ŽFig. 2A. with a rapid onset, a pronounced decay in the continued presence of the agonist, and a rapid offset upon washoff of the amino acid. The taurine responses also appeared concentration-dependent since there was a trend for the higher concentration to produce slightly larger currents ŽFig. 2B.. For 3 mM taurine, current amplitudes were 60 " 13 pArpF Ž n s 6.; while 10 mM taurine produced 96 " 11 pArpF Ž n s 7.. Taurine can act as an agonist for both strychnine-sensitive glycine receptors and GABA A receptors in recombinant and native systems w15,50x. In order to determine whether either of these neurotransmitter receptors mediated responses to taurine by basolateral amygdala neurons, we applied taurine in the presence of saturating concentrations of either the GABA A receptor antagonist, SR95531 Ž1 mM., or the glycine receptor antagonist, strychnine Ž0.3 mM. w16x. Surprisingly, currents gated by 3 mM taurine Ž n s 6. were significantly Žrepeated measures ANOVA. depressed by strychnine Ž22 " 5 pArpF vs. control values of 60 " 13 pArpF, P F 0.01., but not by SR95531 Ž52 " 13 pArpF vs. control, P G 0.05; Fig. 2B1 ., representing 60 " 6% and 12 " 6% inhibition ŽFig. 2B 2 . by strychnine and SR95531, respectively. Similarly, responses to 10 mM taurine Ž n s 7. were also significantly inhibited by strychnine Ž63 " 5 pArpF vs. control values of 96 " 11 pArpF, P F 0.01; 33 " 5% inhibition.. Importantly, 1 mM SR95531 also significantly suppressed the 10 mM taurine response Ž67 " 10 pArpF vs. control, P F 0.01; 30 " 8% inhibition.. For all neurons tested with 10 mM taurine, the inhibitions by 1 and 3 mM SR95531 were 25 " 5% Ž n s 12. and 25 " 8% Ž n s 5., respectively, suggesting that the inhibitory effects of 1 mM SR95531 were very close to maximal. The concentration–response relationship for taurine ŽFig. 3A. yielded an apparent EC 50 s 600 mM and a slope s 1.9 ŽFig. 3D–F; n s 7–9.. Importantly, inclusion of 10 mM bicuculline with the various concentrations of taurine did not significantly influence either the maximal taurine response nor the taurine concentration–response relationship Ž30 mM–3 mM; data not shown., consistent

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Fig. 2. Taurine-gated currents in acutely isolated basolateral amygdala neurons are mediated primarily by strychnine-sensitive receptors. ŽA. Examples of currents gated by different concentrations of taurine in two different isolated neurons. In these traces, the lower concentration of taurine Ž3 mM, A 1 . was relatively insensitive to the GABA A receptor non-competitive antagonist, SR95531 Ž1 mM., while being substantially more sensitive to inhibition by the glycine receptor antagonist, strychnine Ž0.3 mM.. In contrast, the higher concentration of taurine Ž10 mM, A 2 . was substantially inhibited by both SR95531 and strychnine. Holding potential: y60 mV. Calibration bars: for the traces in ŽA 1 ., y s 0.3 nA, x s 2 s; for ŽA 2 ., y s 0.3 nA, x s 1 s. ŽB. Summary of SR95531 and strychnine effects on taurine-gated currents expressed as current density ŽB1 . and percent inhibition ŽB 2 .. The relatively small effect of SR95531 on 3 mM taurine-gated currents Ž n s 6. and the apparent increase in its effect at higher concentrations of taurine Ž10 mM, n s 7. indicate that GABA A receptors significantly contribute to taurine-gated currents only at higher ŽG 3 mM. taurine concentrations, while strychnine-sensitive glycine receptors appear to be primary mediators of taurine-gated currents at concentrations - 3 mM. ‘‘U ’’ and ‘‘UU ’’ indicate values significantly different from control Žrepeated measures ANOVA. at P - 0.05 and P - 0.01 ŽTukey–Kramer multiple comparison post-test., respectively.

with the notion that taurine responses at concentrations F 3 mM are not mediated by GABA A receptors. Together, these results indicate that: Ž1. taurine-sensitive ligand-gated ion channels are expressed by adult basolateral amygdala neurons; Ž2. these channels are predominantly strychninesensitive receptors when using F 3 mM taurine; and Ž3. GABA A receptors may contribute to the taurine currents at concentrations ) 3 mM. 3.2. Pharmacologic characterization of strychnine-sensitiÕe receptors expressed by adult BLA neurons The pharmacology of strychnine-sensitive taurine-gated channels was determined to establish whether these receptors were functionally related to the glycine receptors expressed in the brainstemrspinal cord w6,44x. Current responses to the amino acid, glycine, were found in almost every BLA neuron examined Ž n s 78 in various experiments. and were similar to those initiated by taurine,

although maximal responses to a saturating concentration of glycine Ž1 mM; 255.9 " 53.5 pArpF; see Fig. 7. were substantially larger than those found for the highest concentration of taurine tested Ž10 mM; 96 " 11 pArpF; see Fig. 2.. At moderate concentrations of glycine Ž) 50 mM., the currents were characterized by rapid onset, moderateto-substantial desensitization in the continued presence of agonist, and rapid deactivation once agonist was removed. Furthermore, glycine exerted a concentration-dependent effect on resting membrane current ŽFig. 3B., with an apparent EC 50 s 47 mM and n H s 1.2 ŽFig. 3D–F; n s 5– 6.. In addition to taurine, strychnine-sensitive glycine receptors are also sensitive to an additional beta-amino acid, b-alanine w5,15,40x. Because b-alanine can act as an agonist at GABA A receptors w5,15x, it was applied as an admixture with the GABA A receptor antagonists, bicuculline methiode Ž10 mM. or SR95531 Ž3 mM.. b-Alanine caused concentration-dependent alterations in resting mem-

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Fig. 3. The agonist pharmacology of isolated basolateral amygdala neurons is consistent with the expression of strychnine-sensitive glycine receptors. ŽA–C. Examples of currents elicited by different concentrations of glycine ŽB. and the b-amino acids, taurine ŽA. and b-alanine ŽC.. Holding membrane potential: y60 mV. Calibration bars: ŽA. x s 2 s and y s 0.6 nA; ŽB. x s 1 s and y s 0.6 nA; and ŽC. x s 2 s and y s 0.2 nA. ŽD. Concentration–response relationships generated from fits to the Hill equation for glycine ŽB, n s 5–6., taurine Ž Ø , n s 7–9., and b-alanine Že, n s 5–6.. The effects of a given agonist were normalized to a maximally efficacious concentration of glycine Ž1 mM. and plotted as percent control vs. log 10 wconcentrationx. Note that the concentration–response relationship for b-alanine was obtained in the presence of the GABA A receptor antagonist, bicuculline methiodide Ž10 mM.. The taurine concentration–response relationship was determined both with and without bicuculline methiodide Ž10 mM.; and, because no significant effect of bicuculline was noted, the data from different groups were pooled to give the relationship shown here. ŽE and F. The concentration–response relationships shown in ŽD. yielded the following EC 50 ŽE, log 10 " S.E.M.. and Hill slope ŽF. values Žmean " S.E.M..: for taurine, EC 50 s 600 mM and n H s 1.9; for glycine, EC 50 s 47 mM and n H s 1.2; and for b-alanine, EC 50 s 284 mM and n H s 0.9.

brane current in isolated BLA neurons ŽFig. 3C., yielding an EC 50 s 284 mM and n H s 0.9 Ž n s 5–6; Fig. 3D–F.. In cultured hippocampal neurons w18x, b-alanine acting at GABA A receptors has an EC 50 that is substantially larger Ž4–5 mM. than at BLA glycine receptors reported here. It is therefore likely that the use of GABA A receptor antagonists in these experiments would be sufficient to block any b-alanine activation of GABA A receptors. To conclusively demonstrate that amygdala glycinegated currents were mediated by typical glycine receptors, we tested strychnine sensitivity of glycine-mediated re-

sponses. Because strychnine has been described in some instances as a competitive antagonist for glycine receptors Žreviewed in Ref. w34x., we tested the effects of strychnine on ; EC 50 concentrations of glycine. When applied with glycine as an admixture, strychnine appeared to have the largest effect on apparent current desensitization kinetics rather than the maximal current amplitude ŽFig. 4A.. However, these characteristics were completely altered when cells were exposed to strychnine alone prior to application of the glycinerstrychnine admixture. One possible explanation for these findings, a slow association between

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Fig. 4. Strychnine-mediated inhibition of glycine-gated responses: influence of concentration and exposure time. ŽA. The inhibition of glycine Ž50 mM. current by strychnine Ž0.3 mM. is influenced by prior exposure to the antagonist. In these sample traces, application of a glycinerstrychnine admixture resulted in a marginally depressed peak current amplitude and profound increase in apparent desensitization rate. Pretreatment Ž) 30 s. of the neuron with strychnine prior to application of the admixture completely attenuated the peak current while maintaining the absolute amount of inhibition during the steady state component. Holding membrane potential: y60 mV. Calibration bars: x s 2 s and y s 0.2 nA. ŽB. The effects of strychnine pretreatment are the result of a slow association between antagonist and receptor. After application of 50 mM glycine and allowing the current to reach steady state, rapid application of a glycinerstrychnine Ž0.3 mM. admixture results in an exponential decrease in current amplitude. The time constant Žt . in this example is ; 2 s, indicating that strychnine binding to the glycine receptor reaches equilibrium only after several seconds. For all neurons tested in this way Ž n s 4., t s 1.9 " 0.3 s. Calibration bars: x s 2 s and y s 0.05 nA. ŽC. Concentration–response relationship for strychnine inhibition of glycine-mediated currents. Data collected from different sets of neurons were normalized to the currents elicited by 50 mM glycine Ž100%. and expressed as percent control. For the ‘admixture’ curve ŽB, n s 3–7., peak current amplitudes were used to calculate the percent control values, yielding a concentration–response relationship with an estimated IC 50 s 0.9 mM and n H s y0.5. Strychnine pretreatment Ž%, n s 4–8. resulted in a substantial leftward shift in the concentration–response relationship with the IC 50 s 37 nM and n H s y0.9. ŽD. Increasing times of exposure to strychnine increase its apparent potency. Using current traces resulting from application of the glycinerstrychnine admixture Žno antagonist pretreatment., strychnine IC 50 values were calculated using the mean current amplitude from 100 ms bins of time following the peak current amplitude. For example, the bin labeled ‘0.5 s’ was calculated from the mean current amplitude during the 401–500 ms following the peak of the response. The logŽIC 50 . " S.E.M. were taken from fits of this data and portions are shown here.

strychnine and its receptor, was tested by application of a glycinerstrychnine admixture during the ‘plateau’ phase of glycine-mediated current ŽFig. 4B.. The onset of strychnine-mediated inhibition could be fitted by a single-component exponential function, with an average time constant, t , of 1.9 " 0.3 s Ž n s 4., indicating that in these neurons, it takes several seconds for the strychnine–receptor interaction to reach equilibrium. Regardless, strychnine inhibited current responses to 50 mM glycine in a concentration-dependent fashion ŽFig. 4C.. In those neurons where a glycinerstrychnine admixture was applied without prior strychnine exposure, an estimation of the complete strychnine concentration–response relationship using peak current responses yielded an IC 50 f 0.9 mM Ž n H s y0.5, B

in Fig. 4C; n s 3–7.. However, glycinerstrychnine admixture IC 50 values calculated from mean current amplitudes during successive 100 ms time bins following the peak amplitude increased with time ŽFig. 4D., suggesting an increase in apparent potency with increasing time of exposure to the antagonist. This suggestion is supported by the observation that pretreatment with the antagonist for ca. 30 s prior to application of the glycinerstrychnine admixture further increased the apparent sensitivity to strychnine ŽIC 50 s 37 nM, n H s y0.9, n s 4–8; % in Fig. 4C.. Together, these results strongly suggest that the strychninerreceptor interaction is relatively slow in reaching equilibrium. In summary, pharmacologic analysis of glycine-gated currents strongly suggests that BLA neurons

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express ‘classic’ strychnine-sensitive glycine receptors that are the likely mediators of taurine ŽF 3 mM.-gated responses. 3.3. The glycine current–Õoltage relationship If BLA glycine-gated currents are indeed mediated by ‘classic’ anion-selective channels, the major permeant ion should be chloride. To examine this, the current–voltage relationship for glycine Ž50 mM.-mediated currents ŽFig. 5A. was determined using different intracellular chloride concentrations Ž132 vs. 22 mM.. This concentration of glycine was chosen in order to insure accurate measurement of the peak current amplitude and to avoid the prolonged channel desensitization that can occur following repeated applications of high concentrations of agonist. Neurons were held at test membrane potentials for approximately 15–30 s prior to application of glycine. Approximately linear current–voltage relationships were found Ž n s 4–5. for neurons dialyzed with either 132 mM Cly ŽB, Fig. 5B. or 22 mM Cly Ž', Fig. 5B.. The apparent reversal potential for these glycine-gated currents was estimated by linear regression of current vs. voltage plots and was determined to be approximately y5 mV for neurons dialyzed with 132 mM Cly and y46 mV for neurons dialyzed with 22 mM Cly. These data are in good agreement with the calculated Nernst equilibrium potentials Žy5 mV for 132 mM Cly i and y51 mV for 22 mM . Cly . Thus, glycine-activated currents in isolated basolati eral amygdala neurons are likely to be accounted for by increases in chloride permeability. 3.4. Comparison of GABA- and glycine-mediated currents Previous studies on synaptic transmission in the basolateral amygdala w33x indicate that these neurons receive substantial GABAergic innervation from local interneurons. Consistent with this, we find that application of GABA to isolated neurons produced significant concentration-dependent changes in resting membrane current ŽFig. 6A.. Hill plots of this data Žnot shown. indicate a concentration–response relationship with an EC 50 s 6 mM and slope s 0.9 Ž n s 3.. Thus, despite the relative insensitivity of taurine-gated currents to both SR95531 and bicuculline at concentrations F 3 mM ŽFig. 2., isolated basolateral amygdala neurons express significant GABA-mediated responses. Although glycine and GABA A receptors share many aspects of their pharmacology, it is possible to distinguish these receptors based upon their antagonist sensitivity. Approximately EC 50 concentrations of GABA Ž10 mM; Fig. 6A. and glycine Ž50 mM; Fig. 3B and D. were tested for sensitivity to ‘selective’ concentrations of strychnine Ž0.3 mM. and bicuculline methiodide Ž10 mM. w42x. As shown in Fig. 6B, 300 nM strychnine Žfollowing a ; 30 s pretreatment. preferentially inhibited the glycine-gated currents Ž91 " 4% inhibition, n s 8. compared to GABA-

Fig. 5. Chloride is the predominant permeant ion for glycine-gated currents. ŽA. Sample traces demonstrating the effects of membrane potential and intracellular chloride concentration on glycine Ž50 mM.mediated responses. All records are with wClyxouts159.5 mM. With the wCly x i s132 mM ŽA 1 ., the glycine current reversal potential can be estimated from a linear regression of current amplitudes corresponding to the q20, 0, and y20 mV holding potentials to be y0.4 mV. In a different neuron dialyzed with 22 mM Cly ŽA 2 ., the reversal potential was similarly estimated to be y41.8 mV. Calibration bars: for ŽA 1 ., x s 2 s and y s 0.3 nA; for ŽA 2 ., x s1 s and y s 0.3 nA. ŽB. Summary of current–voltage relationship data for basolateral amygdala neurons dialyzed with 132 mM Cly ŽB, ns 4–5. or with 22 mM Cly Ž', ns 5.. Data are expressed as a current density ŽpArpF. to reduce the influence of cell-to-cell variability. The reversal potentials for each dataset were estimated from the x-intercept of linear regressions Ždashed y lines. and are y5 mV for 132 mM Cly in and y46 mV for 22 mM Cl in . Note that both relationships can also be described as slightly outwardly rectifying although estimations of the reversal potential based upon such non-linear relationships Žy2 mV for 132 mM Cly in and y40 mV for 22 . did not substantially differ from the reported values based mM Cly in upon a linear relationship.

mediated responses Ž18 " 7% inhibition, n s 4.. Data describing strychnine inhibition of glycine are taken from Fig. 3D and are shown here to aid comparisons between treatments. Conversely, 10 mM bicuculline methiodide significantly attenuated GABA-gated currents Ž84 " 4%

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inhibition, n s 6. while having little effect on glycine responses Ž7 " 4% inhibition, n s 7.. These results, along with those presented for the antagonist sensitivity of taurine currents, support the conclusion that basolateral amygdala neurons functionally express strychnine-sensitive glycine receptors that can be distinguished from GABA A receptors. Finally, while these experiments indicate that strychnine-sensitive glycine receptors are indeed expressed by adult rat BLA neurons, it is unclear whether these receptors significantly influence the neurophysiology of BLA neurons. As an initial attempt to address this issue, we compared the maximal current amplitude of glycine-gated responses to those gated by GABA A receptors in isolated neurons. Using maximally efficacious concentrations of both GABA Ž300 mM; see Fig. 6. and glycine Ž1 mM; see Fig. 3., the current densities for glycine- and GABA-gated responses were 255.9 " 53.5 and 317.6 " 29.6 pArpF, respectively ŽFig. 6C; n s 8.. Although the mean glycine responses were ; 81% of the mean GABA-mediated responses, these apparent differences did not reach statistical significance Žpaired t-test, P s 0.11.. Thus, glycine and GABA A receptors are functionally expressed at similar levels.

4. Discussion

Fig. 6. The functional levels of glycine receptor expression by basolateral amygdala neurons are similar to that of GABA A receptors. ŽA. GABAmediated responses are concentration-dependent. The GABA concentration–response relationship Žnot shown. can be described by an EC 50 s6 mM and n H s 0.9. Calibration bars: x s1 s and y s 0.2 nA. ŽB. GABAand glycine-gated currents in isolated neurons can be pharmacologically differentiated. ; EC 50 concentrations of GABA Ž10 mM. and glycine Ž50 mM. were applied along with strychnine Ž0.3 mM. and bicuculline methiodide Ž10 mM.. For the strychnine data, neurons were pre-treated with the antagonist for ca. 30 s before application of the agonistrstrychnine admixture. Note that the glycinerstrychnine data shown here have been re-plotted from the data presented in Fig. 4 in order to aid in comparisons between treatments. The glycine-gated currents were substantially inhibited by strychnine Ž91"4% inhibition, ns8. but not by bicuculline methiodide Ž9"2% inhibition, ns 7.. Conversely, GABAmediated currents were sensitive to bicuculline methiodide Ž88"2% inhibition, ns6. but substantially less sensitive to strychnine Ž18"7%, ns 4.. ŽC. Functional glycine and GABA A receptors are expressed in similar levels. Responses to maximally efficacious concentrations of glycine Ž1 mM. and GABA Ž300 mM. were compared by normalization to measured cell capacitance Ž ns8.. Average responses to 1 mM glycine were 256"54 pArpF while those to 300 mM GABA were 318"30 pArpF.

In this study, we have demonstrated that isolated adult rat basolateral amygdala neurons respond to extracellular application of glycine and the b-amino acids, taurine and b-alanine, with pronounced changes in resting membrane current. Like GABA A and ionotropic glutamate receptors, relatively high concentrations of ‘native’ agonists are required to produce maximal responses from amygdala glycine receptors. For these ‘amino acid’-gated receptors, it is likely that such high neurotransmitter concentrations are achieved at the synapse where postsynaptic receptors and presynaptic release sites are precisely juxtaposed. The concentrations of glycine needed to produce maximal responses in isolated basolateral amygdala neurons Že.g., G 300 mM. are quite similar to those needed for maximal strychnine-sensitive glycine receptor activation in cultured rat spinal w44x and cerebellar granule neurons w47x, in isolated neonatal rat forebrain neurons w1,41,42x, in recombinant systems expressing individual human glycine receptor subunits w30,40x, and in zebrafish hindbrain neurons w19x. The low potency of native ‘amino acid’ agonists at strychnine-sensitive glycine receptors is therefore characteristic of these ligand-gated chloride channels. While taurine is known to act as a partial agonist for GABA A receptors, pharmacological characterization of taurine-gated responses in isolated adult basolateral amygdala neurons indicates that they are likely to be mediated by strychnine-sensitive glycine receptors. This conclusion is supported by a pronounced sensitivity of taurine concen-

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trations F 3 mM to strychnine and a lack of antagonism by the GABA A receptor antagonist, SR95531. Incomplete inhibition of the responses to 3 mM taurine by strychnine is likely a reflection of the competitive nature of the inhibition Žreviewed in Ref. w34x., although an additional non-competitive component may also be involved in the strychninerglycine receptor interaction w1,51x. The competitive nature of the strychninerglycine receptor interaction may also be reflected by our findings that 10 mM taurine is proportionally less sensitive to strychnine than are lower taurine concentrations. However, because the sensitivity of 10 mM taurine to SR95531 is proportionally greater than for lower taurine concentrations ŽFig. 2., it is likely that concentrations of taurine greater than 3 mM may also activate GABA A -mediated chloride conductance. Taken together, these findings indicate that a concentration of taurine in the range of 30 mM–3 mM is likely to activate predominantly strychnine-sensitive glycine receptors. Because the taurine and b-alanine currents produced by concentrations F 3 mM were likely mediated solely by strychnine-sensitive glycine receptors, both currents were normalized to currents activated by 1 mM glycine in order to facilitate their comparison. As can be seen from the concentration–response relationships in Fig. 3D, taurine was not as efficacious as glycine Ž3 mM taurine was 70% of maximal glycine response., and was therefore likely to be a partial agonist at BLA glycine receptors. Taurine is also a partial agonist for recombinant human glycine receptors expressed in Xenopus oocytes and is less efficacious at homomeric glycine receptors composed of a 2 subunits compared to those composed of a 1 subunits w40x. Although this might suggest that BLA glycine receptors are composed of a 2 subunits, it is difficult to compare the pharmacological properties of native and heterologously expressed channels due to a number of factors, including potential differences in post-translational modification. Furthermore, it is likely that amygdala glycine receptors contain the b subunits w11,20x, and that inclusion of the b subunit has been shown to significantly alter some aspects of receptor pharmacology w30,36x. The subunit composition of amygdala glycine receptors awaits more detailed investigation. The strychnine sensitivity of BLA glycine receptors also deserves some comments. Similar to reports on glycine receptors in isolated neonatal neurons from the ventral tegmentum w51x, the inhibition of glycine currents by strychnine takes several seconds to develop in adult amygdala neurons. Because strychnine can apparently bind to receptors prior to application of an agonist ŽFig. 4A., our findings are consistent with those of Ye et al. w51x in that strychnine does not act solely on the open channels. Although not directly addressed in this study, the slow dissociation of antagonist from the receptor may be indicated by a ‘relaxation’ away from baseline at the onset of agonistrstrychnine application following strychnine pre-

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treatment ŽFigs. 2A 1 and 4A.. Our findings, that the apparent potency of strychnine increased during continuous exposure to a glycinerstrychnine admixture ŽFig. 4D., are also consistent with a relatively slow unbinding of strychnine compared to glycine w24x, provided that strychnine binds to BLA glycine receptors in a competitive fashion. Nonetheless, because several studies have indicated that strychnine inhibition of glycine receptors may depend upon both competitive and non-competitive components and that electrophysiological approaches neces-sarily derive information from a number of complex, allosteric interactions, it may be unwise to estimate association or dissociation rates Žand subsequently calculate K d . from exponential fits of data like that shown in Fig. 4. The apparent IC 50 for strychnine reported here Ž37 nM. is similar to values reported by a number of studies using neonatal neurons from spinal cord w44,49x and forebrain w1,41,42x as well as adult hypothalamus w16x. Although it is well-established that glycine-gated chloride currents can be found in neonatal forebrain neurons w1,41,42x, our results indicate that strychnine-sensitive glycine receptors can also be found in the limbic forebrain of adult rats since the age of the rats used in this study was significantly greater than 4 weeks Žsee Section 2.. Although the exact role of these glycine receptors in the neurophysiology of the basolateral amygdala remains to be determined, available evidence indicates that there is a relative paucity of glycine-positive terminals or fibers within the limbic forebrain w35x, suggesting that BLA glycine receptors may not be activated by synaptically released glycine Žbut see Refs. w7,26x.. However, BLA neurons themselves appear to concentrate intracellular taurine w28x, perhaps suggesting that strychnine-sensitive glycine receptors expressed by these neurons may play some sort of ‘regulatory’ role. This possibility could shed some light on the potential behavioral consequences of taurine release in the basolateral amygdala in response to stressors like ethanol w31x or elevated extracellular Kq w10x. Although taurine has been postulated to play numerous cellular roles w17x, the potential ‘non-synaptic’ regulation of a neurotransmitter receptor by this amino acid is an exciting alternative to more classically defined roles for ligand-gated ion channels. Given that facilitation of GABA A receptor function in the basolateral amygdala by benzodiazepine agonists can profoundly attenuate anxietyrfear-related behaviors w38x, stressor-induced taurine release in this same brain region may act in a similar fashion but via activation of strychnine-sensitive glycine receptors. Acknowledgements This work was supported, in part, by a Research Starter Grant from the Pharmaceutical Research and Manufacturers of America Foundation. We thank Drs. G.D. Frye and J.T. Trezciakowski for their helpful comments.

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