Brain Research, 594 (1992) 115-123

115

© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

BRES 18200

Cl--mediated interaction between G A B A and glycine currents in cultured rat hippocampal neurons F r a n c e s c a Grassi Department of Experimental Medicine; Unii'ersityof L 'Aquila, and Laboratory of Biophysics, Regina Elena Cancer Institute, Rome (Italy) (Accepted 26 May 1992)

Key words: ),-Aminobutyric acid; Glycine; Hippocampal neuron; Chloride equilibrium potential

A cross-interaction between GABA- and glycine-evoked currents was found when the two transmitters were applied in sequence to cultured embryonic rat hippocampal neurons. Whole-cell GABA-current was inhibited by a previous glycine-current flowing in the same direction (inward or outward), and potentiated by a current with opposite polarity. The same effect was caused by GABA on glycine-current. Repeated applications of GABA (glycine) elicited currents of decreasing or increasing amplitude, according to a similar pattern. Transmitter interaction was independent of external Ca 2 + and of all the metabolic pathways tested, but it was blocked by specific receptor antagonists, bicuculline and strychnine. The extent of both inhibition and potentiation correlated with the amount of charge flowing through the membrane during the conditioning transmitter application, indicating that cross-modulation depends on shifts of CI- reversal potential. This finding has both functional and methodological implications, as it suggests a new mechanism of transmitter interaction in the brain, and also that patch-clamp pipettes cannot adequately perfuse cell interior.

INTRODUCTION Glycine (GIy) and y-aminobutyric acid (GABA) are the two main inhibitory neurotransmitters in the central nervous system. Two different subtypes of GABA receptors (GABAAR and GABABR) i°, and one type of GIy receptor (GlyR)Thave been described. Both GABAAR and GIyR contain an integral anionic channel, and belong to a superfamily of ligand-gated ion channels, together with nicotinic acetylcholine (AChR) and glutamate receptors. All these receptors are oligomeric proteins carrying the transmitter binding sites, and forming the ion ch~.nnel7. The ability of neurotransmitters and neuropeptides to alter the response of receptor-channels gated by other transmitters plays a key role in regulating neuronal electrical activity. Modulation of receptor function is controlled through pathways involving GTP-dependent proteins (G proteins) and second messengers. For example, receptor desensitization, which takes place when receptors are continually exposed to their transmitters, has been reported to be regulated by

several kinase systems, both for the muscle nicotinic AChR, the best studied model 12'~'~,and for some neuronal receptors 1"~'21.Some examples of neuromodulators acting directly on transmitter receptors are also known. Gly binds to the N-methyI.D-aspartate (NMDA) type of glutamate receptors, potentiating cell response to the excitatory transmitter ~''~'~. Similarly, GABAAR carries benzodiazepine binding sites, whose occupation can up- or down-regulate chloride current H~. Cross.inhibition of the currents evoked by ionophoretically applied GABA and Gly was observed in mouse spinal neurons 4, but the mechanism of the interaction remained unclear, though GABA and Giy were reported to be able to alter local chloride concentration in hippocampal ~4, spinal and cerebellar neurons 5. In cultured embryonic rat hippocampal neurons, a reversible cross-interaction between the two inhibitory neurotransmitters was consistently found: the amplitude of whole-cell GABA-evoked current was modified by a previous Gly application, and vice-versa GABA regulated Gly-evoked current. However, each transmitter acted only on its own receptor, ruling out the

Correspondence: F. Grassi, Laboratory of Biophysics, Regina Elena Cancer Institute, via delle Messi d'Oro 156, 1-00158 Rome, Italy. Fax: (39) (6) 4180473.

116 possibility that this effect is analogous to the modulation of NMDA receptor by Gly. Since both GABAand Gly-induced currents were able to shift the chloride equilibrium potential, the possibility that transmitter cross-interaction was due to local changes of CIconcentration was examined.

Mannheim) were added, just prior to use, to the patch-pipette solution from stock solutions at 100 mM (in water), and tested up to 30 rain form the rupture of the membrane patch, l-(5-1soquinolinylsulfonyl)-2-methyl-piperazine (H-7) dihydrochloride (Sigma) was dissolved in water at 30 raM. it was applied to the neurons for up to 20 rain, either in the perfusion medium (100/~M) or intracellularly, via the patch pipette (100 or 300/zM). Forskolin (Sigma) was stocked in ethanol at 10 mM and then added to the external solution (final concentration: 10 #M). Each substance was tested independently, in at least four cells internally dialyzed with CsCI-solution.

MATERIALS AND METHODS

Eiectrophysiological recordings and data analysis Cell cuhure Primary cultures of hippocampal neurons were prepared as follows. Hippocampi were dissected from the brains of 17- to 20-day-old Sprague-Dawlcy rat embryos. Cells were mechanically dissociated and plated in culture dishes (35 ram, Falcon) coated with low molecular weight poly-L-lysine (15,000-30,000, Sigma), dissolved (1 mg/ml) in H3BO3/Na.IB4Ov buffer (4.76 g/I and 2.54 g/I, respectively). Polymer was added to the dishes (1.5 rag/dish) and incubated for 30 rain under a sterile hood (about 28°C). The dishes were then rinsed twice with distilled water and dried. Seeding density was approximately calculated as the yield of 1.5 hippocampi/dish. Culture medium was Dulbecco's minimum essential medium (DMEM) modified according to Kaufman and Barrett Is (I.5 ml/dish), and it was partially exchanged every 10 days. Cells were incubated at 370C, with 5% CO 2. Neuronal processes formed an extended network within the first days in culture. Cells survived up to 4 weeks. Transmitter-evoked currents increased in amplitude during the Ist week, and remained stable thereafter. Experiments were performed on neurons cultured for 8-22 days, with comparable results. Cell age was therefore neglected in subsequent data analysis.

Solutimrs and la,rfusion During experimenls, the culture medium was replaced by a normal external solution (NES) containing (in mM): 140 NaCI, 2.8 KCI, 2 CaCIz, 2 MgCl~, 10 glucose, l0 HEPES/NaOH, ptl 7.3. In few experiments, CaCI, was replaced by BaCI~. GABA and Gly (both Sigma) were added to NES from frozen stock solutions 100 times more concentrated than the final transmitter concentration, which was 10 ~M for GABA and I(XI/~M for Gly, unless otherwi;e stated. Tetrodotoxin (0.5/~M, Sigma) was added to all solutions, to block synaptic transmission, in some experiments, GABA was replaced by either muscimol or baclofen (both Sigma), as indicated. Transmitters were administered to the neurons via a gravity-driven perfusion system, A three-barrelled pipette, placed 100-300 ~tm from the cell, was connected to three reservoirs, containing NES, GABA and Gly solution, respectively. Opening and closing of each perfusion line was controlled by hand-operated stopcocks. During recordings, cell were continuously superfused with control solution. Transmitter responses were evoked by switching to superfusion with either the GABA- or Gly-containing solution, Care was taken to switch the two stopcocks simultaneously. Perfusion efficacy was estimated by the rate of current rise and by the fall of current upon transmitter removal. When the time necessary to reach half of maximal current amplitude exceeded 500 ms, the recording was discarded. A similar consideration applied to the wash-out phase. Different salt solutions were used to fill recording pipettes. One ('CsCI-solution') contained (in raM): 120 CsCI, 20 tetraethylammo. nium chloride (TEA-O), I CaCI 2, 2 MgCI 2, II EGTA, 10 HEPES/ TEA-OH, 2 Na-ATP; pH, 7.3. To improve recording conditions, it was generally diluted by 20% To record in low CI- conditions, two different solutions were used. Solution 'CsFa' contained, in raM: 120 CsF, 0.5 CaCI 2, 1.5 MBCI2, 10.5 EGTA, I0 HEPES/TEA-OH, 2 Na-ATP; pH, 7.3. Solutiou "CsFb' contained, in raM: 100 CsF, 20 CsCI, 0.5 CaCI 2, 1.5 MgCI,, 10.5 EGTA, 10 HEPES/TEA-OH, 2 Na-ATP; pH. 7.3, Intracellular solutions were stocked at -20°C in I ml aliquots and used within 2 weeks, so that ATP (Boehringer-Mannheim) degradation was still negligible, as determined by HPLC analysis (data not shown). GTPyS (l(g} or 500 ~.M) and GDP/3S (500 p.M) (Boehringer-

All the experiments were performed at room temperature (25°C) using the whole-cell recording technique. Transmitter-evoked currents were recorded from the cell soma by borosilicate glass pipettes (2-5 MK] resistance), filled with one of the intracellular solutions (see above), and connected to a List EPC-7 amplifier. Patch series resistance (Rs), estimated by compensation circuit, was generally below 7.5 MD. During recordings, the cell holding potential was generally clamped at - 8 0 or - 9 0 inV. When necessary, the holding potential was shifted to the desired value just prior to transmitter application, then shifted back to the original level following transmitter wash-out. Data were digitized at 100 Hz and stored on a computer (Schneider 386) for subsequent analysis. Evaluation of current amplitude and decay rate was carried out using AUTESP programs, kindly provided by H. Zucker (Max Planck Institute for Psychiatry, Germany). The peak current amplitude was measured as the distance from the baseline, taking into account all the shifts due to variations of cell membrane holding potential. To minimize artefacts due to inadequate voltage clamp, only the responses not exceeding 5 hA, from recordings in which Rs was lower than 5 ME2, were fitted to determine the time course of current decay, so that potential errors at the peak of current response were of 25 mV at most. With these cautions, the larger currents had a time course consistent with that of the smaller ones. Results are given as mean + S.E.M..

RESULTS Current inhibition In most neurons, both GABA and Gly, when separately applied (see Methods for details on perfusion system), evoked current responses (lo^ua and Ioi~, respectively), that were measured using the whole-cell variation of the patch-clamp recording technique. The amplitude of IoauA and of lol ~ varied from cell to cell, depending, among other factors, on transmitter concentrations, and on the driving force for chloride ions. Using CsCl-solution (see Materials and Methods), at the cell holding potential (HP) of - 9 0 mV, applications of both GABA (I0 IzM) and Gly (I00 IzM) triggered inward currents with comparable peak amplitude, ranging from 0.5 to I0 nA (Fig. la and Table I). When CI- in the internal solution was partially substituted with F- (OF-solutions), the inward GABA- and Gly-elicited currents reverted at HPs more positive than - 7 5 mV (Fig. Ib and Table I). If two transmitter applications (2-5 s) were separated by at least 2 min wash, current responses remained stable or decreased by less than 10% (see for an example Fig. 2). On the contrary, repeated transmitter applications at short time intervals (less than I0 s) elicited currents of decreasing amplitude ('self-inhibition'). When GABA (or

117 Gly) was administered twice, with 1 s interval between applications, current amplitude decreased by about 50%, as indicated in Table I (see also Fig. 6). Consecutive applications of GABA and Gly caused the modification of the evoked currents ('cross-inhibition'), as illustrated in Fig. lb. If GABA was administered before Gly, IGty resulted smaller than the corresponding control current. In turn, ]GABA was reduced with respect to the control, following the application of Gly. The current inhibition was fully reversible after prolonged wash (longer than 1 min). Cross-inhibition was observed with all intracellular solutions, at any membrane potential tested, in the range - 1 3 0 to - 2 0 mV (Fig. lb,c). After Gly (100 ~M) applications lasting 1.5-90 s, the current evoked by GABA (10 ~M) was around 50% of the control, independent of the duration of the conditioning transmitter application. At the same concentrations, following GABA treatments of comparable duration, IGly was around 40% of the corresponding 'untreated' currents (see Table I for values in various experimental conditions). No significant difference in the extent of current reduction was found when GABA concentration was changed by 10folds, while Gly was used at 1 mM (data not shown). Gly reduced the current evoked by 10 ~M GABA to 49 + 8% (n = 4), and that elicited by 100/~M GABA to 60% (n - 2) of control. In parallel, IGty was reduced to 51 + 7% (n = 4) of control by 10 ~M GABA, and to 58 + 5% ( n - 3) by 100 ttM. This dose.independence st~ggests that the inhibition of treated current amplitude was not due to a decrease of receptor affinity for its transmitter.

Current cross-inhibition persisted even when a brief wash was interposed between the two transmitter applications. The influence of a conditioning perfusion lasting 2-5 s gradually vanished over several seconds, as shown in Fig. 2a. During this wash, some cells were briefly hyperpolarized by 70 mV, then depolarized again, with no effect on GABA-GIy interaction (not shown). This demonstrates that voltage steps do not interfere with current modulation and provides a control for subsequent experiments. Pharmacology To test whether cross-inhibition was due to the direct action of GABA on GlyR and of Gly on GABAAR, the effects of bicuculline, a specific blocker of GABAAR, and of strychnine, a specific blocker of GIyR, were examined. When the cells were perfused with a solution containing GABA (10/~M) plus bicuculline (10 /zM), /GABA was blocked (Fig. 2b, right panel) and Gly-activated current was not affected (4 cells) (Fig. 2b). In the same way, the Gly-containing solution (100/zM) additioned with strychnine (1 ~M) did not elicit current response (Fig. 2c, right), and was ineffective in reducing IGABA(4 cells, Fig. 2c). These data indicate that each transmitter must act on its own receptor to condition the current evoked by the other. The possible involvement of GABABR was ruled out by the fact that muscimol (10/zM), a specific agonist of GABAAR, elicited large currents, and induced the reduction of Gly.activated response to 31 + 1% (n - 3) of the corresponding control value. Muscimol-elicited currents were reduced by Oly to 59 + 13% of control

TABLE !

Amplitude of transmitter.induced currents and effects of t~arious conditioninR treatments Currents evoked by GABA (10/~M) and Gly (100 ,tiM), recorded using the different internal solutions (int. sol,), in control conditions and after various treatments. Currents conditioned by GABA or by Gly, as indicated, were measured as percent of the corresponding control. Positive amplitudes: outward currents, measured at - 5 0 mV and at - 10 mV for CsFa- and CsFh-sOlution, respectively. Negative values: inward currents, measured at - 9 0 mV (CsCI- and CsFb-sOlutions) or at -120 mV (CsFa-solution). Perfusion protocols were as follows: self.inhibition wa,-, obtained by two applications of the same transmitter with 1 s interval; cross.inhibition was induced by applying the two transmitters in sequence; self-and cross-potentiation were measured with 2 s interval between transmitter applications. In all the experiments, the conditioning perfusion was maintained till the transient current component had subsided, in parentheses: number of cells, nd: not determined,

Int. sol.

CsCI CsFa CsFa CsF b CsFb

Control amplitude

Self.inhib.

Self.potent.

Cross.inhib.

(n,,I)

(%)

(%)

(%)

/GABA -5.6 +0.6 (16) +0.53+0.06(19) -0.524-0.05(18) +1.7 4-0.3 (3) -2.6 4-0.6 (6)

(1) (5) (I) (1)

l~ty CsCI CsEa CsE~ CsF b CsF b

-4.1 4-0.4 (16) +0.384-0.05 (10) -0.564-0.05 (7) +1.164-0.15 (4) -2.3 4-0.4 (7)

(%)

Giy

by GABA 74 51+6 47 nd 36

nd 137+ 9 (6) 2054-36 (7) nd 132 (1)

55+8(13) 47+3(11) 504-6 (8) nd 554-9 (5)

nd 136 200 nd nd

454-5 364-5 33+4 18+9 48+4

by Gly nd 62 28 nd 46

(1) (1) (2)

Cross.potent.

nd 138+ 9 (6) 144 (2) nd 132 (1)

by GABA (1) (1)

(5) (9) (4) (4) (5)

nd 1584-21 (4) 1534- 8 (7) nd 140 (1)

118 (n = 3 ) ( d a t a not shown). Furthermore, baclofen (10 lzM), a specific agonist of G A B A . receptor, was totally ineffective on lcly (3 cells). Neither G A B A A R nor G l y R are considered as G protein-coupled receptors. It is therefore unlikely that the interaction between G A B A and Giy is mediated by metabo;ic pathways, as it was confirmed by experiments in which the effects of drugs that influence some cell signalling mechanisms were examined. H-7, a non-

HP=-90 mV a

GABA

Gly

a..

90

30

I

,

0

b

I

i

i

5 interval (s)

GABA~Bicuculline

Gly

10

Gly _

I ,,,,

,,,

q

2nA

4s

GABA

4s

b Gly GABA

m

GABA

GABA

HP=-50 nlV GABA Gly W

W

0,2hA I

Oly

Gly+stryehnine

W

2s

HP..120 mV GABA

Oly W

Fig, I, Typical GABA- and Gly-evoked currents in embryonic hippocampal neurons and their cross-interaction, a: currents evoked by GABA (left,) and by Gly (right) in a neuron internally perfused with CsCI-solution at HP = - 90 inV. In this cell, currents were best fitted by two exponential components, according to the equation: Ao + Ate-~/'+ A . e -~/'', The theoretical curves, superimposed to the traces, are: I~|ABA = -- 1.0Q- 1.55 e - ~ / ~ ' 3 - !,52 e-t/t'2; IGtw ~ - 1,16 - 1.61 c-t/?~ _ 1.4e4e-~/t 4 (1" in s, coefficients in hA). b: GABA-Gly cross-inhibition in another neuron, internally perfused with CsF~. ~)lution CliP --- -50 mV), The left panel shows the control (C) Italy and the treated (T) !(;^,^. The right panel shows the control IGABA and the treated /t;ty. The peak amplitude of the Gly-treated lt3AnA is reduced to 33% of control (right trace), while GABA-treated Italy is 23% of its control Cleft panel), c: a similar perfusion protocol was applied, in the same neuron as in b, at HP -- - 120 mV. Note that in this trace Gly-treated lt~^.^ (44% of control) lacked the exponentially desensitizing phase, l~ty was reduced to 39% of control by GABA treatment. The interval between consecutive recordings was 3 rain. Bars above the traces indicate the perfusion protocol. Hatched bars: wash (W).

Fig, 2, Persistence and pharmacology of GABA-Gly interaction, a: GABA-Gly cross-inhibition gradually vanishes when the two transmitter applications (each lasting 2-5 s) are separated by a wash of increasing duration. The amplitude (relative to control) of Gly-treated i(t^,^ (o) and of GABA-treated I¢,ly(.) is plotted vs, the duration of the interval between consecutive applications, Data from a single neuron, dialyzed with CsFt, solution, Recordings were repeated every 2 rain, b: btcuculline (10 ~tM) blocks both GABA-induced current and inhibition of icily, in another neuron, Control I(jl~ (left panel) is practically identicai after conditioning application of GABA :rod bicuculline, which elicits only a very small residual current (right trace), Perfusion as indicated by bars above traces (wash omitted in this figure). Membrane potential was -90 mV, with CsCI-solution in the patch pipette, c: strychnine (I pM) blocks both Gly-evoked current and the inhibition of IGAnA. GABA-evoked current is the same in control conditions (left) and after treatment with Gly and strychnine (right panel). Recording conditions as in b, in a different neuron. Bars show the perfusion protocol,

specific protein kinases inhibitor, GTP,/S and GDP~S, respectively activating and inhibiting all G-proteins, and forskolin, an activator of adenylate cyclase, were all unable to affect the cross-interaction between G A B A and Gly (data not shown; see Methods for details on drugs application). In addition, this interaction persisted also when CaCI2 in the external solution was replaced by BaC! 2 (2 cells) indicating that the presence of Ca 2+ ions was not required for current reduction to develop. Current potentiation

Since cross-inhibition was observed in both inward and outward currents, experiments in which IOAnA and

119 IGly had opposite directions (Fig. 3) were performed. This was achieved by shifting membrane potential between GABA and Gly applications, which were generally separated by a brief wash (see Fig. 3), to reduce spurious conditioning currents. In most neurons, an outward GABA-current was evoked first, followed by an inward Gly-current (Fig. 3a, left). After washing for 2-4 rain, the currents were evoked in the reverse sequence (Fig. 3a, right). Following Gly treatment, the amplitude of the outward 16AriA increased to 140% of control value (Table I). The intensity of the GABAtreated inward IGly was enhanced to 160% (Table I) of the control. A similar 'cross-potentiation' was also observed for inward IGAaA and outward 161~(Table !). As cross-inhibition mimics the effect of multiple transmitter applications, it was of interest to test whether also the enhancement of 161y could be ina GABA

Gly I

c

Gly

GABA

-50mY

T

0 . 4s 1nA

~ a , j c q

GABA

-60mV -70mV

m

-5 -70mV

I -70mV

Fig. 4. Current potentiation restores the decaying component of IGAnA. a: in this neuron, internally perfused with CsFa-solution, close to Eci (about - 6 5 mV), the inward lC~ABA virtually lacked the decaying component Ai, and maintained the same amplitude during repeated applications, b,c: conditioning outward currents restored the decaying component of the inward 16An,x, whose amplitude increased with that of the conditioning stimulus. Note that, once the transient component had subsided, /GABA had the same amplitude as in a. Voltage and perfusion protocols are indicated under the traces (wash omitted).

duced by Gly itself, using a modified version of the protocol described above. An outward lcly was followed, after a brief wash, by an inward IGly and vice versa (Fig. 3b). Both inward and outward currents were potentiated to about 150% of control (Table I). A comparable 'self-potentiation' was induced by GABA on la^,^ (Table i and Fig. 4).

Kinetics of current decay

b

~

c

"---

-120 mV

4S

Gly

b

Gly

Gly

" - " -120

•

-,'-'

Gly I

.50mY

-'--

mV

Fig. 3. Current potentiation is induced by transmitter-evoked currents flowing in opposite directions, a: GABA-Oly interaction. Treated (T) laAn^ (right trace) is 153% of control (C) response (left panel), while treated laly (left trace) is 143% of the corresponding control (right). Consecutive recordings, separated by 2.5 rain wash, from a neuron internally dialyzed with CsF,-solution. Voltage and perfusion protocols are indicated above the traces. Wash is omitted in this figure. Membrane potential was always shifted during the wash between transmitter applications. Note that a transient current was evoked by residual GABA upon membrane hyperpolarization, which increased the driving force for CI- ions. b: Oly.induced potentiation of iat ~. Outward-treated Icily increased to 132% of control (right and left trace respectively). Inward current was potentiated (left) to 194% of control (right). Consecutive recordings with 2.5 rain interval, from the same cell as a. Note, upon membrane hyperpolarization, the same transient as in a. The whole experiment lasted 1 h, and the mutual transmitter interaction was detected over the whole period.

During sustained transmitter application, GABAand Oly-evoked currents invariably declined (e.g. Figs. 1-3), except when the cell holding potential was less than 10 mV more negative than Eel (Fig. 4a). The decaying phase of I(;ABAand iaty was best fitted by the sum of a constant component plus an exponentially decaying one, according to the equation: !~ = A 0 + Ate -t/~'. In control conditions, the constant component (A o) accounted for about 30% of the GABA-current, and for about 20% of the Gly-current (see Table 11). The remaining fraction of the signal (,4 i) decayed as a single exponential with time constant (I-) ranging from 0.5 to 3 s (Table II). in some cells, if transmitter application outlasted 10 s, a second exponential component with greater ~" could be evidenced (see for example, Fig. la). Multiple transmitter applications elicited currents in which only the decaying component (A I) consistently differed from the control, while A o and ~" were generally unaffected. Both cross- and self-modulation (inhibition and potentiation) obeyed to this rule, as summarized in Table 111. Occasionally, cross-inhibition totally abolished the decaying component, resulting in

120 TABLE II

Parameters of the best fit of transmitter-induced control currents Transmitter-induced control currents were best-fitted with the equation ,4 o + Ate~/': the ratio A = A o / ( A o + Al) represents the contribution of the constant component to the total current. Recordings showing two decaying components, or shorter than 2~" are not included. For each internal solution used, current direction is indicated by arrows: 1' for outward, ,[ for inward. Membrane potentials and transmitter concentrations as in Table I. In parentheses: number of cells.

Int. soi

GABA ~'¢s)

CsCIi 2.2 -+0.4 CsFaT 1.25±0.19 CsF a J, 0.87±0.10 CsFt,1' 1.34_+0.16 CsFh I 0.79±0.06

Glycine T(s)

A

30±3 (17) 2.4 4-0.3 24±3 (19) 1.49_+0.36 32_+3 (18) I.II_+0.14 41:1:5 (3) 1.32_+0.03 30_+2 (6) 0.77±0.08

A 12:1:2 17± 2 17± 2 14_+ 6 13± 12

(16) (13) (9) (4) (7)

flat transmitter-evoked currents (e.g. Fig. lc, left). Conversely, a potentiating outward current added a transient component to non-decaying inward control currents elicited near the reversal potential (three cells). The amplitude of the decaying component was proportional to the conditioning stimulus (Fig. 4b,c), and no self-inhibition was observed in the flat currents (Fig. 4a). These data strongly support the hypothesis that only the decaying component of IGAaA and !¢31y is modulated, while the constant component of the current can be maintained for tens of seconds.

Rob, of CI ~.concentration The most straightforward explanation of the observed current inhibition/potentiation phenomenon is that, in spite of intracellular dialysis with the saline solution in the patch pipette, the conditioning current causes local changes of Cl- concentration, thus modifying the driving force during the subsequent treated

current. This explains both the symmetric effect of GABA and Gly, and the cross-interaction being independent of the cell metabolism. GABA-induced shifts of Eo in neuronal cells have been previously reported 2,5,~4. To test this working hypothesis, a detailed investigation of the cross-interaction between ]GABAand Icly was carried out around the current reversal potential. These experiments were performed using the CsF-solutions, so that E a was relatively negative, and the activation of voltage-dependent channels was negligible. With 6 mM internal CI- (nominal concentration in CsFa-solution), both /GABA and [Gly w e r e biphasic when the membrane-holding potential was very close to the putative E o of about - 6 0 mV (Fig. 5a,b). This behaviour is consistent with the assumption that currents are able to shift [CI-] i, since at HP close to the reversal potential, the driving force mainly depends on the ion concentration gradient, and it is therefore very sensitive to local changes in CI- concentration. The biphasic behaviour was less marked when the concentration of CI- in the patch pipette was raised to 20 mM (CsFt,-solution), so that small changes in [CI]i were better 'buffered'. With this solution, the reversal potentials were - 3 5 + 1 mV and - 3 4 + ! mV for/GABA and Ioy, respectively (n = 10). Fig. 5 shows that conditioning currents were able to shift current reversal potential. The extent of the shift depended on the direction and on the amplitude of the conditioning current (Fig. 5c-e), as it was observed in all the ten cells tested. Under the present working hypothesis, the extent of current inhibition (potentiation) should be related to the amount of CI- ions accumulated (extruded) during the conditioning transmitter application, that is to the integral of the conditioning current. To look for this

TABLE I!!

Effect of different condilioning treatmoll~' on best fit parameters of l¢iaRA and ltil~, Conditioned currents were best fitted with the equation Ao + A le ' / ' , The parameters are given as percent of the corresponding control (see Table !1). nd: not determined, Values indicated by (*): the difference with the control (100%) is significant at the 0,01 level (Student's t-test). All the results obtained for inward and outward currents, with the various intracellular solutions, were pooled, as not significantly different. Membrane potentials and transmitter concentrations as in Table I.

GABA

Glycme

A, et-~; Self-inhibition 78±10 .

.

.

.

.

.

.

.

.

.

.

.

Self-potentiation 107± 8 Cross-inhibition 91)± 6 Cross-potentiation 147± 12 *

,~

Aj t%)

1"(%;

Ao (%7

At t%)

¢ (%)

41-+ 6 "

!!6-+9 (6)

nd

nd

nd

172_+ 17 *

122:1:7(!1)

78

40± 5 *

110+9(36)

86± 9

37-+ 3 *

127± 12 (31)

140:!: 9 *

90±5 (8)

109±21

158± 12 *

iO! + 4 (9)

160

96

(2)

121 correlation, a protocol similar to that illustrated in Fig. 3 was used. Conditioning currents (2-5 s) were evoked at different cell membrane potentials, in the range - 140 to - 10 mV, so that different amounts of charge crossed the membrane. Test transmitter applications were invariably performed at - 9 0 mV, 2 s after the conditioning treatment. Since most of the data collected indicate that only the decaying component of the current is involved in the inhibition (potentiation) phenomenon, the constant component A 0 was sub=

a

GABA W

-35mY I

..,

b

-90 mV

Gly

W .11./h

a i

',

',

",i

j

///;

Gly ii

I SOpA

-90 mV

-35mV "//I

[~ -90 mV _

b

GABA

~

'~,"r- f,

~

'-

=

.

-62mY

1$

d -33mV

-33mV

-90mV

GABA

e -lOmV

[

-33mY

GABA

.,..,...._

i 0,SEA

f

leo

~

140

~

(I

• 0

120 1B ~-- 100 O0 60

,= •1

o conditioningcha~e (nO)

1

Fig. 5. Transmitter-evoked currents are able to shift Ecl. a,b: currents evoked by OABA (a) and Gly (b) are biphasic, when cell holding potential is near £cl, and internal CI- concentration is low. Both traces are from a neuron internally perfused with CsF,,-solution. The broken lines indicate the baseline. Note the expanded vertical scale, c-e: with higher [CI- ] in the patch pipette (CsFh-sOlution), at HP = - 3 3 mV control GABA.applications elicit virtually no current (c). At the same potential, IOAnA becomes outward following a conditioning inward current (d). By contrast, an inward IGAeA is elicited after a conditioning outward current (e). Traces recorded from the same cell, with 2 rain interval. Voltage and perfusion protocols are indicated above the traces, f: the extent of current inhibition/potentiation well correlates with the charge accumulated/extruded by the transient conditioning pulse. The peak amplitude (as percent of control) of the treated current (HP = - 90 mV) is plotted vs. the charge (in nC) flowing across the cell membrane during the conditioning transmitter applications (lasting 2-5 s), performed at different potentials ( - 140 to - 10 mV), to obtain a range of inhibitions and potentiations. Results from two neurons (CsF bsolution): (e): GABA-treated IGiy; regression coefficient 0.994. (o): Gly-treated I~AnA; regression coefficient 0.985. Inset: only the decaying component of the conditioning current (hatched region) was integrated. Interval between transmitter applications: 2 s.

Fig. 6. Receptor desensitization alone does not explain self-inhibition. a, left: at the cell membrane potential of - 9 0 mV, the current elicited by the second GABA-application is about 50% of that elicited by the first application, a, right: at - 3 5 mV, GABA elicits a very small current, but still desensitizes its receptor. The current evoked by the second transmitter application (HP = - 9 0 mV) is about "/0% of the control (left panel), b: similar effects are induced by Gly, in a different neuron. Voltage and perfusion protocols are indicated above the traces.

tracted from the signal to be integrated (see inset in Fig. 50. A linear relation between the effect and the integral of the transient conditioning current was found in both self- and cross-modulation (5 cells), with correlation coefficients greater than 0,98 (Fig. 50, while no correlation was found when the total conditioning current was considered, Since self-inhibition of IO^,A and lc,~ was largely, comparable with the cross.inhibition, an attempt was made to establish the relative role of receptor desensitization and of shifts of E a in controlling the decrease of current amplitude. When cell membrane potential is very close to E a , transmitter applications evoke virtually no current, but still cause receptor desensitization. As shown in Fig. 6, GABA or Gly administered at cell HP close to Ecj reduced the current elicited by a second transmitter application ( H P - - 9 0 mV) to about 70% of the control. For comparison, conditioning applications with similar duration but at HP - - 9 0 mV reduced the test current to about 30% of the control. This suggests that receptor desensitization and changes of [Cl-]~ have similar importance in determining current decay. Such significant shifts of Ec~ are striking, as they imply that intracellular dialysis with patch pipette solution does not control the composition of the intracellu-

122 lar milieu. This point was further confirmed by experiments carried out in the outside-out configuration, which allows full perfusion of the inner membrane side. In all the three excised patches examined, IGABA and lo~y were readily evoked by transmitter application, indicating good recording conditions. However, no obvious cross-interaction was observed when the two transmitters were applied in sequence (data not shown), since the peak amplitude of both currents remained constant. DISCUSSION This paper provides evidence that, in cultured embryonic hippocampal neurons, the consecutive activation of GABAAR and GlyR induces cross-modulation of the two transmitter-elicited currents, due to changes of chloride equilibrium potential. IOAaA and lomy are either inhibited or potentiated by conditioning currents with the same or opposite polarity, respectively. The transmitter cross-modulation is blocked by specific antagonists for GABAAR (bicuculline) and GlyR (strychnine), indicating that each transmitter acts only at its own receptor. This is confirmed by the fact that muscimol, a specific agonist of GABAA R, mimics the action of GABA, while baclofen, a specific agonist of GABAaR, has no effect on ioly. GABA and Gly can change chloride driving force in several neuronal preparations 2''~'~4''~, potentially affecting all the currents carried by CI =. The data here reported show that chloride redistribution induces transmitter cross-interaction, and provide an additional proof that, during sustained exposure to the transmitters, the current response of GABAAR and GlyR is independently modulated by receptor desensitization, which invariably leads to current depression, and by shifts of intracellular chloride concentration, which potentiate or depress the transmitter response. For example, repeated GABA (Gly) applications, known to result in inhibited currents, can also lead to potentiated /GABA (ic;~y), if current polarity shifts during transmitter superfusion. Several pieces of evidence indicate that only the decaying phase of the response is involved in the CI--mediated modulation. The extent of inhibition or potentiation depends linearly on the amount of charge flowing during the transient part of the conditioning current, but shows no correlation with the charge flux carried by the steady-state current. This point is also confirmed by the observation that the duration of the conditioning application has no effect on cross-modulation, once the transient current has subsided. Moreover, current inhibition and potentiation selectively suppress or restore the decaying component of the

response. It is as if the hippocampai neurons can only 'buffer' changes of [Cl-] i at a low rate. The large current which develops upon transmitter application induces a rapid accumulation or extrusion of CI-, which cannot be compensated for by the neuron. So, [Cl-] i continues to change till ECI reaches such a value, that current flow is compatible with the neuron buffering ability, and a steady charge flow can be maintained. This component may be modulated by receptor desensitization. Therefore, the fast decay phase invariably observed in IGABAand IGly might be due to changes of [Cl-] i, while the slower component, only occasionally resolved, might represent the contribution of receptor desensitization, and this model would he in accordance with the observations of Huguenard and Alger 14 in adult hippocampal neurons. The response of receptors of the same superfamily to sustained transmitter applications seems to be differentially regulated. For example, the desensitization of the muscle nicotinic AChR is accompanied by an increased rate of decay of the response ~7, while conditioned /GABA and lo~~ never decayed faster than the controls. The present findings contrast with the report by Barker and McBurney 4 that the depression of inhibitory amino acid responses in spinal neurons is not due to shifts of Eo. Moreover, the many reports that the stimulation of protein kinase systems reduces 2"~'24 or increases ~l'~s neuronal response to GABA and Gly applications should take into account the possibility that the target of the phosphorylation reaction is not the receptor, but some other protein(s) involved in the control of [C! ° ]i. Consistent with the hypothesis that the local concentration of Cl- plays a key role in transmitter interaction, no cross-inhibition takes place when the intracellular medium is fully controlled by the recording pipette, as in outside-out patches. By using the suction pipette recording technique, which provides a better intracellular perfusion than the standard whole.cell pipette, changes of E o in bullfrog sensory neurones occurred on a much longer time scale 2 than that here reported, and no interaction between GABA and GIy was found in freshly dissociated adult rat ventromedial hypothalamic neurons 27. By contrast, when using classical intracellular recording techniques, both cross-inhibition of IOAaA and loly and transmitter-induced changes of the local chloride concentration were observed in central neurons 4. Changes of [Cl-] i are likely to be physiologically relevant to neuronal integrated activity, since it was reported 2~' that repetitive activation of GABAergic synapses modifies the reversal potential of I~ABA"It is very likely that shifts of E o mediate cross-talking

123 between GABA and Gly also in rive. It was in fact observed that GABA-containing terminals can be apposed to GlyR in rat spinal neurons 2a. Such a co-localization of GABAergic and glycinergic synapses might occur also in other neuronal types, since it is now becoming evident that Gly acts as a neurotransmittcr not only in the spinal cord 9, but also in higher regions of the central nervous system. Responses to Gly have been observed in hippocampal slices of rats during the first two weeks of postnatal life tS, though they later disappear. In adults, GlyRs have been identified in the brain t, and in particular in the hippocampus a. It has also been reported that neuronal response to GABA and Gly can be either excitatory or inhibitory, depending on both the cell resting potential and Ec~, in neonataP '~5 as well as in adult preparations 3,22. Thus, the self- and cross-potentiation of transmitter-evoked currents might take place during normal neuronal activity, if during a GABAergic or glycinergic discharge, an excitatory stimulation intervenes that depolarizes the cell membrane potential to values more positive than E a. In conclusion, two mechanisms of transmitter interaction at postsynaptic sites have been proposed up to now, as reviewed by Kupfermann2°: transmitters allosterically act on the ligand-gated receptor activated by other transmitters. Alternatively, transmitters, binding to G.protein-associated receptors, trigger either membrane-bound or cytosolic processes that result in the functional modulation of ligand-gated channels. The data here presented suggest that the redistribution of the ion flowing through ligand-gated channels underlies a novel mechanism for transmitter interaction, possibly relevant to synaptic plasticity. Acknowledgements. ! am very grateful to Dr. F. Eusebi for continuous advice and valuable suggestions during this work. i thank Drs. S. Alema, C. Caratsch, T. Takahashi and E. Wanke for critical reading of the manuscript, Dr. D. Ragozzino for help with cell cultures and F. Galli for technical assistance. This work was supported by a grant of Ministero Universit,~ e Ricerca Scientifica e Tecnologica and by a FIDIA grant (to Dr. F. Eusebi).

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