Neuron, Vol. 10, 83-88, January, 1993, Copyright 0 1993 by Cell Press

Enhancement of p-Adrenergic Responses by Gi-Linked Receptors in Rat Hippocampus

Rodrigo Andrade Department of Pharmacological

and Physiological Science St. Louis University School of Medicine St. Louis, Missouri 63104

Summary

Many excitable cells express a class of neurotransmitter receptors functionally defined by their ability to increase potassium conductance through C proteins of the Gil G, class that directly activate the potassium channels. Biochemical studies have shown that these same recep- tors can also inhibit forskolin-stimulated adenylyl cy- clase, although the functional significance of this effect remains unclear. In this study electrophysiological tech- niques were used to examine how activation of serotonin and y-aminobutyric acid receptors belonging to this class affect g-adrenergic responses signaled through adenylyl cyclase. Surprisingly, activation of these receptors not only failed to inhibit but actually enhanced g-adrenergic responses. These observations are consistent with evi- dence indicating that G-linked receptors can enhance the ability of C, to stimulate certain adenylyl cyclases.

Introduction

Electrophysiological studies havecharacterized afam- ily of neurotransmitter receptors that act through G proteins(Andradeetal.,1986)oftheGi/G,class(Simon et al., 1991) to elicit a membrane hyperpolarization and inhibit excitable cells (Nicoll et al., 1990; Birn- baumer et al., 1990). Previous work in several systems has shown that this effect is mediated by a class of inwardly rectifying potassium channels whose activa- tion is signaled by a direct interaction between the G protein and the ion channel (Pfaffinger et al., 1985; Andradeet al., 1986; Kurachi et al., 1986). Interestingly, parallel biochemical studies have shown that these receptors are also capable of inhibiting adenylyl cy- clase. This raises the question of the possible func- tional roleofthesechanges in cyclic nucleotide levels. ln particular, does the ability of these receptors to in- hibit adenylyl cyclase allow them to reduce signaling through G,-adenylyl cyclase-CAMP? To address this question I have determined the effects of 5-hydroxy- tryptamine 1 (5-HT,) and y-aminobutyric acid B (GABAtJ receptors, two receptor subtypes known to increase potassium conductance and inhibit adenylyl cyclase, on physiological responses signaled through the G,- adenylyl cyclase-CAMP signaling cascade in single py- ramidal neurons of the rat hippocampus.

Results

Intracellular recordings were obtained from pyrami- dal cells of the CA1 region in in vitro rat brain slices.

Previous studies have demonstrated that administra- tion of norepinephrine (NE) to these cells results in a decrease in the calcium-activated afterhyperpolariza- tion (AHP) present in these cells (Madison and Nicoll, 1986a; Haas and Konnerth, 1983) (Figure IA). This re- sponse is mediated by the activation of 8-adrenergic receptors, which in turn stimulate adenylyl cyclase, increase intracellular CAMP, and signal the reduction of this afterpotential (Madison and Nicoll, 198613). This well-established 8-adrenergic response in the hippo- campus provides an avenue for testing the possible functional significance of the regulation of cyclic nu- cleotides by 5-HT1 and GABAe receptors at the level of a single neuron. Therefore we first examined whether stimulation of 5-HT, receptors modulated the ability of NE to signal a reduction in the AHP. As previously reported (Andrade and Chaput, 1991) administration of 5-carboxyamidotryptamine (5-CT, 300 nM) elicited a membrane hyperpolarization that was associated with an increase in membrane conductance (Figure IA). This hyperpolarization reflects the activation of 5-HTIA receptors by 5-CT in these cells (Andrade and Nicoll, 1987; Andrade and Chaput, 1991). As could be expected from an indirect effect of the increase in membrane conductance, the AHP amplitude was de- creased by the administration of 5-CT. Nevertheless, when NE (0.1-I PM) was administered in the presence of 5-CT, its ability to reduce the AHP was significantly enhanced (Figure 1). This enhancement in the ability of NE to reduce the AHP was observed in all 6 cells tested. Overall, in this group of cells 5-CT increased the effect of NE by approximately 50%, from a mean reduction in AHP amplitude of 3.5 mV, observed when NE was applied alone, to 5.5 mV, when NE was admin- istered in thepresenceof 5-CT(p<O.OOl, paired ttest). This enhancement in the effect of NE was observed in spite of the increase in conductance, which would work against observing such an increase. To compen- sateforthe5-CT-induceddecreasein membraneresis- tance, the effects of NE were normalized with respect to the AHP amplitude immediately preceding its ad- ministration (Figure IC). Under these conditions 5-CT was found to enhance the ability of NE to reduce the AHP, from 26% to 53% (p < 0.001; Figure 2A).

Receptors of the ~-HTIA and GABAt, subtypes in the rat hippocampus hyperpolarize pyramidal cells of the CA1 region through a shared mechanism that results in the activation of acommon subpopulation of potas- sium channels (Andrade et al., 1986). Therefore it was of interest to determine whether activation of the GABAe receptors in these cells could also potentiate B-adrenergic responses. As previously reported (New- berry and Nicoll, 1984; Andrade et al., 1986), adminis- tration of the selective GABAe agonist baclofen (IO- 30 PM) resulted in a membrane hyperpolarization. In addition, as seen with 5-CT, concurrent with the hy- perpolarization there was a significant enhancement in the ability of NE to reduce the AHP. In this group

A 5-CT WkM NE 1gM -

B Control

In 5-CT

Reduction of the AHP Figure 1. Effect of 5-CT on the B-Adrenergic-Induced

(A) Administration of 1 PM NE to this cell reduced the amplitude of the AHP (downward deflections) elicited in this cell by a single calcium spike (upward deflection) by 33%, from 13.5 mV to 9 mV. Administration of 300 nM WIT, a concentration that selectively stimulates 5-HTIA receptors in these cells (Andrade and Chaput, 1991), elicited a 7.5 mV membrane hyperpolarization. Notice that during the hyperpolarization the depolarizing pulse failed to reach threshold for the calcium spike, and hence there was no AHP. After current injection was used to compensate for this hyperpolarization, a second administration of NE reduced the AHP by 82%, from 8.5 mV to 1.5 mV. After removal of the 5-CT from the bath, the cell membrane potential gradually recovered to control. A final administration of NE now reduced the AHP by 42%, from 13 mV to 7.5 mV. The asterisks denote selected AHPs depicted in (B) using an expanded time scale. The enhancement of the NE response by 5-CT is best appreciated by superimposing expanded AHPs obtained under control conditions and in the presenceof NE before (left) and during (middle) theadministration of 5-CT. Rightmost traces depict AHPs obtained in the presence of 5-CT but scaled as to match the amplitude of the control (i.e., pm-NE) AHPs. The graph in (C) plots the time course of the reduction in the AHP by NE in the presence and absence of 5-CT. Closed circles, NE administered alone; closed squares, NE administered in the presence of S-CT, closed triangles, NE administered after recovery from the 5.CT. Cell membrane potential, -70 mV.

C -J

NE alone

Y-T-=-- I NE’“5-CT

-2 -1 0 1 2 3 4 5 6

Time (min)

A 100

0

8 /4 .A

-;-/ . .

. / P

. . 8 0

Control in 5-CT

B 80

0

Figure 2. Comparison of the Effects of 5-CT and Baclofen on the Ability of NE to Reduce the AHP

i . . 4 . l

. b l

Control In Baclofen

(A) 5.CT (300 nM) enhanced the ability of NE (0.1-I wM) to elicit a reduction in the AHP. (B) Similar effects were seen in 7addL tional cells tested with baclofen (lo-30 PM). In (A) and (B) the effects of NE on the AHP are expressed as percentage of control, which takes into consideration that 5-CT and baclofen increase membrane conduc- tance and, therefore, indirectly reduce the amplitude of the AHP. However, it is note- worthy that in most cases (11/13) these ago- nists also enhanced the absolute ampli- tude of this reduction, in spite of the fact that the AHP itself was smaller than under control conditions.

Enhancement of &Adrenergic Responses 85

In Baclofen I’ r !k-

Recovery

I+=

I- L---

i[ /--- ii Control and IS0

1

b ,I?--

/

m$roo;~~ so ,,z KS0

1'

Figure 3. Baclofen Enhances the Ability of lsoproterenol to Re- duce the AHP

Top row, effect of isoproterenol (ISO) on the AHP following a calcium spike. Each AHP was preceded by a hyperpolarizing constant current pulse to assess simulataneously membrane in- put resistance. Second row, administration of baclofen (IO PM) elicited a 7 mV hyperpolarization, which was compensated by using +dc current to restore the membrane potential to control. As previously reported this hyperpolarization was associated with an increase in membrane conductance, which appeared to account in this cell for most of the reduction in the AHP ampli- tude. The ability of 2 nM IS0 to reduce the AHP was markedly enhanced in the presence of baclofen. Third row, 20 min after removal of the baclofen from the bath, membrane potential and the ability of IS0 to reduce the AHP have recovered to control. Bottom trace, superimposed AHPs obtained under control con- ditions and in the presence of ISO. Rightmost panel illustrates control and IS0 AHPs in the presence of baclofen, but they are scaled to match the amplitude of the AHP in the absence of baclofen. Cell membrane potential, -71 mV.

of cells 30 PM baclofen enhanced the ability of NE to reduce the AHP from 14% to 36% (n = 7 cells tested, p < 0.005, Figure 26). As with 5-CT, the enhancement of NE by baclofen could be seen independent of the normalization of the data. Thus in this group of cells baclofen enhanced the absolute magnitude of the p-adrenergic response by approximately 75%, from a mean reduction in AHP amplitude of 1.9 mV to 3.4 mV (p < 0.01, paired t test). Similar effects were observed when the selective B-adrenergic agonist isoproterenol (l-100 nM) was used instead of NE to stimulate G, (Figure 3). Under these conditions baclofen still elic- ited a significant enhancement in the ability of this agonist to reducetheAHP(n = 4cellstested, p<O.O5).

Baclofen and serotonin could enhancefl-adrenergic responses either by increasing the ability of the recep- tor to activate adenylyl cyclase or by acting more dis- tally to magnify the effect of CAMP once generated. To distinguish between these possibilities, the ability of baclofen to enhance the effect of the membrane permeable CAMP analog 8-bromo-CAMP was exam- ined. As illustrated in Figure 4, baclofen (30 PM) failed

to enhance the ability of 8-bromo-CAMP (300 nM) to reduce the AHP (n = 4 cells tested, p = 0.7). In three of these experiments it was possible to compare in the same cell the effects of baclofen on the ability of NE and &bromo-CAMP to reduce the AHP. Although baclofen failed to enhance the ability of 8-bromo- CAMP to reduce the AHP (22% versus 23%, p = 0.9), it nevertheless significantly enhanced that of NE (from 16% to 53%, p < 0.05, Figure 4).

In these experiments 5-CTand baclofen elicited two distinct responses: they hyperpolarized thecell mem- brane by increasing potassium conductance (New- berry and Nicoll, 1984; Andrade and Nicoll, 1987), and they enhanced the ability of fl-adrenergic receptors to signal a decrease in the AHP. The question could be asked whether the apparent enhancement of the fi-adrenergic response might not be secondary to the increase in conductance that underlies the hyperpo- larization. To test this possibility, we took advantage of the recent demonstration that intracellular injec- tion of the lidocaine derivative QX-314 could block

the 5-CT-and baclofen-induced increase in potassium conductance while leaving the AHP unaffected (An- drade, 1991). As illustrated in Figure 5, injection of QX-314almost completely blocked the ability of baclo- fen or 5-CT to elicit a membrane hyperpolarization. The mean hyperpolarization elicited by baclofen (30 PM) in this group of QX-314injected cells was 1.6 k 0.7 mV (mean f SD), which was significantly different from that elicited under control conditions (II.3 +

In baclofen

Figure 4. Effect of Baclofen on the Ability of 8-Bromo-CAMP to Reduce the AHP

Top row, &bromo-CAMP (0.3 mM for 5 min) elicits a reduction in the AHP. Middle row, administration of baclofen (30 PM) elic- ited a 10 mV hyperpolarization, which was compensated by a passing +dc current through the electrode to restore the mem- brane potential to control level. Baclofen however failed to en- hance the ability of B-bromo-CAMP (0.3 mM for 5 min) to reduce theAHP. In contrast, in the samecell, baclofen (30pM) enhanced the ability of NE to reduce the AHP from 33% to 70% (data not shown). Bottom row, superimposed AHPs obtained under con- trol conditions and in the presence of bbromo-CAMP before and after baclofen administration. Cell membrane potential, -64 mV. The graph on the right summarizes the results from 3 cells in which it was possible to compare on the same cell the effects of baclofen on B-bromo-CAMP and NE responses. The error bars correspond to standard deviations. Asterisk denotes p < 0.05.

NfXlr0ll 86

After QX314 Injection

II Wash

In Baclofen

J/---- L------ J/.--

Control and NE Control and NE Control and NE c? 0

Control In Baclofen

Figure 5. Effect QX-314 on Baclofen Responses

In a cell injected with QX-314, administration of NE (1 PM) reduced the AHP by 34%. Administration of baclofen hyperpolarized the cell membrane from -71 mV to -73 mV (inset). A second administration of NE now elicited a reduction in the AHP amplitude of 72%. Upon removal of the baclofen from the bath the membrane potential gradually recovered, and a final administration of NE reduced the AHP by 33%. Similar results were obtained in 4 additional cells, as plotted in the accompanying graph. Even after QX-314 injection, baclofen still could elicit a variable reduction in the AHP. The mechanism underlying this effect is unclear but might be related to a reduction in calcium influx, since GABA and 5-HTIA receptors are known to reduce calcium currents (Dolphin et al., 1990; Penington and Kelly, 1990). (Inset) Horizontal calibration bar represents 2 min. Vertical calibration is the same as in the main body of the figure.

1.5 mV, p < 0.001). QX-314 injection also significantly reduced the decrease in AHP amplitude associated with baclofen administration but did not completely suppress it. The mean reduction in AHP amplitude elicited by baclofen was 5.5 f 1.7 mV under control conditions and 3.5 + 1.1 mV after QX-314 injection (p < 0.05). In spite of this near total blockade of the increase in potassium conductance, baclofen (30 PM) was still capable of enhancing the ability of NE to reducetheAHP(Figure5).OveralI, inagroupof5cells injected with QX-314, baclofen enhanced the ability of NE to reduce the AHP from 23% to 59% (n = 5 cells, p < 0.001). A similar enhancement of NE responses was seen in 3 cells injected with QX-314 and tested with 5-CT (300 nM).

Discussion

5-HT, and GABAe receptors are representative of a vast family of neurotransmitter receptors functionally defined by their ability to couple to G proteins of the Gi/G, family (Simon et al., 1991) to inhibit adenylyl cyclase and increase potassium conductance. Since the ability of these receptors to increase potassium conductance is independent of changes in cyclic nu- cleotides, one of the outstanding questions regarding this family of receptors is to define the functional role played by the regulation of adenylyl cyclase. To ad- dress this issue the present study examined the effect

of 5-HT, and GABAe receptor activation on B-adrener- gic responses mediated by the G,-adenylyl cyclase- CAMP cascade in single pyramidal cells of the CA1 region. Surprisingly, the ability of B-adrenergic recep- tors to reduce the AHP in these cells was not reduced by 5-CT or baclofen, but instead was enhanced.

How might this potentiation of B-adrenergic re- sponses be mediated? A simple increase in NE avail- ability at the receptor site, secondary to a reduction in uptake, seems unlikely, since responses to isopro- terenol were also potentiated, yet isoproterenol is a poor substratefor neuronal uptakein slices(Hirayama et al., 1991). A second possibility is that the enhance- ment of the B-adrenergic responses might be second- ary to the increase in membrane conductance elicited by the 5-HT, and GABAB receptor activation. This pos- sibility also seems unlikely. Baclofen and 5-CT en- hanced the ability of NE to reduce the AHP even after QX-314 injection, which blocked the Increase in potas- sium conductance. Moreover, an increase in conduc- tance, such as that elicited by these agonists, would have been expected to diminish the absolute magni- tude of the reduction in the AHP. Yet the absolute reduction of the AHP by NE or isoproterenol was larger in the presence of baclofen or 5-CT. Finally, a third possibility is suggested by the observation that G-coupled agonists could still reduce the AHP even after QX-314 injection. If the effect of NE were nonlin- ear, being most pronounced on such reduced AHPs,

Enhancement of 8-Adrenergic Responses 87

this could explain the observed enhancement. How- ever, if such nonlinearity existed, it should have also resulted in an enhancement of the &bromo-CAMP. This was not observed in the present experiments. Moreover, no evidence of nonlinearity could be de- tected upon examination of the effects of 1 VM NE on cells displaying a wide range of AHP amplitudes (8-16 mV, n = 18 cells examined). Thus it appears unlikely that the reduction in the AHP by baclofen or 5-CT observed in the presence of QX-314could account for the enhancement of B-adrenergic responses.

These results therefore suggest that 5-HT, and GABAe receptors most likely enhance B-adrenergic re- ceptor-mediated responses in these cells by facilitat- ing their ability to stimulate adenylyl cyclase. These results are consistent with the previous observation in brain slices that baclofen, while inhibiting for- skolin-stimulated cyclase, actually enhanced the abil- ity of B-adrenergic receptors to generate CAMP (Kar- bon and Enna, 1985; Schaad et al., 1989). A possible molecular mechanism for this enhancement resides in the ability of By subunits liberated by the stimula- tion of G proteins by 5-CT and baclofen to synergize with activated a, to stimulate adenylyl cyclase type II (AC II), as recently reported (Tang et al., 1991; Feder- man et al., 1992). This possibility is consistent with recent in situ hybridization studies reporting the pres- ence of AC II mRNA in the CA1 region (Mollard et al., 1992, Sot. Neurosci., abstract).

From the present results it is not possible to deter- mine whether a single receptor/G protein couples to both potassium channels and adenylyl cyclase or whether these effector mechanisms are regulated by separate receptor/G protein subpopulations. It is pos- sible that a single receptor subtype acting at a single class of G proteins might couple to both adenylyl cy- clase or potassium channels. Alternatively, different receptor subtypes might selectively couple to either of these effecters through one or more G proteins. Previous electrophysiological studies have demon- strated the presence of only a single class of 5-HT, receptor, the 5-HTIA subtype, on pyramidal cells of the CA1 region. This suggests that the enhancement observed in the present experiments might be medi- ated by receptors of the ~-HT,A subtype. However, the possible roleof other membersofthe rapidly growing 5-HT, receptor family cannot be ruled out at this time, especially as some of these other receptor subtypes are also expressed in hippocampus (McAllister et al., 1992; Voigt et al., 1991).

5-HT, and GABAe receptors are part of a large group of receptors defined functionally by their ability to inhibit adenylyl cyclase and increase potassium con- ductance (North et al., 1987). Since the opening of potassium channels is signaled through a mechanism independent of changes in cyclic nucleotides (An- drade et al., 1986), it is unclear how the regulation of adenylyl cyclase contributes to the signaling by these receptors. One possibility is that these receptors might be able to inhibit signaling by receptors acting

through the G,-adenylyl cyclase-CAMP biochemical cascade. Some support for this possibility has been obtained from studies on myenteric neurons (Palmer et al., 1987) and heart cells (Fischmeister and Hartzell, 1986). The present results however suggest that such regulation is not universal. Indeed, in the present study, activation of 5-HTIA and GABAe receptors not only failed to reduce but actually enhanced the ability of B-adrenergic receptors to signal a decrease in the AHP. The existence of two cyclases, one stimulated and the other inhibited by the By subunits of the G proteins (Tang and Gilman, 1991; Tang et al., 1991; Federman et al., 1992), could provide a plausible mo- lecular mechanism for these divergent observations. Thus, in cells expressing predominantly adenylyl cy- clase type I (AC I), Gi-linked neurotransmitter recep- tors might not onlydirectlygate the opening of potas- sium channels but also inhibit signaling through the C&-AC I-CAMP cascade. Alternatively, in cells express- ing predominantlyAC II, G-linked receptors signaling increases in potassium conductance would enhance the action of neurotransmitters acting through G,-AC II-CAMP, as reported here for hippocampal neurons. Although the precise identity of the G protein mediat- ing the actions of 5-HT1A and GABAB receptors in the hippocampus is unknown, this mechanism only re- quires that the appropriate BY subunits be released. The existence of these two mechanisms helps resolve contradictory reports regarding the actions of Gi-linked receptorson adenylyl cyclase in the brain (Karbon and Enna, 1985; Schaad et al., 1989; De Vivo and Maayani, 1986; Wojcik et al., 1990; Markstein et al., 1986). More importantly, they might endow cells with extraordi- nary flexibility in integrating different G protein- mediated inputs.

Experimental Procedures

Hippocampal brain slices were prepared using conventional techniques as previously described (Andrade and Chaput, 1991; Andrade and Nicoll, 1987). Briefly, male Sprague-Dawley rats were sacrificed under halothane anesthesia, and the brains were removed and cooled in oxygenated ice-cold saline. The right and left hippocampi weredissected, and 488 urn slices were cut using either a manual tissue chopper or a vibratome. The freshly pre- pared brain slices were then allowed to recover for at least 1 hr in a static interface chamber at room temperature under a moist atmosphere of 95% O2 and 5% COz. After this period slices were transferred one at a time to a recording chamber where the slice was held submerged and was continuously perfused (I-2 ml/ min) with Ringer’s solution of standard composition (119 mM NaCI, 2.5 mM KCI, 1.3 mM MgS04.7H20, 2.5 mM CaCb, 1 mM NaHzPOc 26.2 mM NaHCOJ, 11 mM glucose, saturated with 95% Oz and 5% CO,). In the recording chamber the slice was main- tained at 30°C f l°C.

Intracellular recordings were obtained using microelectrodes filled with 2 M potasssium methylsulfate and exhibiting resist- ances ranging from 88 to 120 MD. Pyramidal cells of the CA1 region were impaled either by passing a brief high voltage pulse through the electrode or by increasing the capacity compensa- tion of the amplifier to cause the head stage to oscillate momen- tarily. Signals were amplified using an Axoclamp IIA amplifier and recorded on a Gould (model RS 3488) paper chart recorder. QX-314 was dissolved into the electrode solution at a concentra- tion of 50 mM and was injected into the cell by passivediffusion,

Neuron 88

aided in some cases by injection of constant current (0.1-0.3 nA). All experiments were conducted in the presence of 1 PM tetrodotoxin and 5 mM tetraethylammonium. Under these con- ditions a depolarizing current pulse triggers a single calcium spike that is followed by a large AHP. Calcium spikes were elic- ited continuously for the duration of the experiment at a fre quency sufficiently slow to allow full decay of the AHP (usually onceevery30-40s). Effectson theAHPin thetext refertochanges in AHP amplitude. The amplitude of the AHP was defined as the maximal amplitude attained by the AHP following a calcium spike. Drug effects on AHPs were quantitated based upon aver- ages of three control AHPs whose amplitudes were compared with those of two to three AHPs obtained at the peak of the agonist effect. All drugs were administered dissolved at known concentrations. In most experiments the effects of an agonist were compared over repeated applications on the samecell. This was accomplished by always administering the same concentra- tion of agonist for exactly the same length of time, while main- tainingaconstantflow rateintothechamber. Statisticalcompari- sons used two-tailed t tests.

Acknowledgments

This work was supported by grant MH 43985 and the Alfred P. Sloan Foundation. I thank Drs. H. R. Bourne and D. V. Madison for suggesting the 8-bromo-CAMP experiments and Drs. R. A. Nicoll and S. G. Beck for reading the manuscript.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertiseme& in accordance with 18 USC Sec- tion 1734 solely to indicate this fact.

Received July 29, 1992; revised September IO, 1992.

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