GABA Activity Mediating Cytosolic Ca2 Rises in Developing ...GABA Activity Mediating Cytosolic Ca21 Rises in Developing Neurons Is Modulated by cAMP-Dependent Signal Transduction Karl

GABA Activity Mediating Cytosolic Ca21 Rises in DevelopingNeurons Is Modulated by cAMP-Dependent Signal Transduction

Karl Obrietan and Anthony N. van den Pol

Department of Biological Sciences, Stanford University, Stanford, California 94305, and Department of Neurosurgery,Yale University, School of Medicine, New Haven, Connecticut 06520

In the majority of developing neurons, GABA can exert depo-larizing actions, thereby raising neuronal Ca21. Ca21 elevationscan have broad consequences during development, inducinggene expression, altering neurite outgrowth and growth coneturning, activating enzyme pathways, and influencing neuronalsurvival. We used fura-2 and fluo-3 Ca21 digital imaging toassess the effects of inhibiting or activating the cAMP signaltransduction pathway on GABA activity mediating Ca21 risesduring the early stages of in vitro hypothalamic neural devel-opment. Our experiments stemmed from the finding that stim-ulation of transmitter receptors shown to either activate orinhibit adenylyl cyclase activity caused a rapid decrease inCa21 rises mediated by synaptically released GABA.

Both the adenylyl cyclase activator forskolin and the inhibitorSQ-22,536 reduced the Ca21 rise elicited by the synaptic releaseof GABA. Bath application of the membrane-permeable cAMPanalogs 8-bromo-cAMP (8-Br-cAMP) or 8-(4-chlorophenylthio)-cAMP (0.2–5 mM) produced a rapid, reversible, dose-dependentinhibition of Ca21 rises triggered by synaptic GABA release. Po-tentiation of GABAergic activity mediating Ca21 rises was ob-served in some neurons at relatively low concentrations of themembrane-permeable cAMP analogs (20–50 mM). In the presenceof tetrodotoxin (TTX), postsynaptic Ca21 rises triggered by thebath application of GABA were only moderately depressed (13%)

by 8-Br-cAMP (1 mM), suggesting that the inhibitory effects of8-Br-cAMP were largely the result of a presynaptic mechanism.

The protein kinase A (PKA) inhibitors H89 and Rp-39,59-cyclicmonophosphothioate triethylamine also caused a large reduction(.70%) in Ca21 rises triggered by synaptic GABA release. Unlikethe short-term depression elicited by activation of the cAMP signaltransduction pathway, Ca21 depression elicited by PKA inhibitionpersisted for an extended period (.30 min) after PKA inhibitorwashout. Postsynaptic depression of GABA-evoked Ca21 risestriggered by H89 (in the presence of TTX) recovered rapidly,suggesting that the extended depression observed during synap-tic GABA release was largely through a presynaptic mechanism.Long-term Ca21 modulation by cAMP-regulating hypothalamicpeptides may be mediated through a parallel mechanism.

Together, these results suggest that GABAergic activity me-diating Ca21 rises is dependent on ongoing PKA activity that ismaintained within a narrow zone for GABA to elicit a maximalCa21 elevation. Thus, neuromodulator-mediated changes inthe cAMP-dependent signal transduction pathway (activationor inhibition) could lead to a substantial decrease in GABA-mediated Ca21 rises during early development.

Key words: mediobasal hypothalamus; calcium; GABA;GABA excitation; protein kinase A; cAMP; development; digitalimaging

The role of GABA as an excitatory neurotransmitter during theearly stages of neural development has been documented over thepast several years (for review, see Cherubini et al., 1991). Severalstudies have shown that GABA triggers neural excitability byactivating the GABAA receptor (Ben-Ari et al., 1989; Chen et al.,1996). Because of a relatively depolarized Cl2 reversal potential(Chen et al., 1996), opening of the GABAA receptor allows Cl2 toefflux from the neuron, triggering membrane potential depolar-ization, activation of voltage-sensitive Ca21 channels, and, as aresult, an increase in intracellular Ca21 (Yuste and Katz, 1991;Yamashita and Fukuda, 1993; Obrietan and van den Pol, 1995).Although glutamate receives a lot of attention regarding its abilityto elevate cytosolic Ca21 levels in developing neurons, we foundthat many developing neurons show a greater Ca21 elevation inresponse to GABA than to an equimolar concentration of gluta-

mate (Obrietan and van den Pol, 1995). As neurons mature,GABA becomes a predominantly inhibitory transmitter that de-creases cytosolic Ca21 levels (Obrietan and van den Pol, 1995).GABA has been shown to possess many of the effects of other,better characterized, fast excitatory neurotransmitters known toincrease intracellular Ca21 during development. For example,GABAA receptor activation can trigger BDNF induction(Berninger et al., 1995) and alter the neural phenotype (Marty etal., 1996). In addition, DNA synthesis in cortical neural progenitorcells can be blocked by specifically inhibiting GABAA receptoractivity (LoTurco et al., 1995). GABA can increase Ca21 levels inneurites and growth cones (Obrietan and van den Pol, 1996a),which may alter growth cone motility and the rate of neuriteextension. In addition, GABA-mediated Ca21 influx in growthcones leads to an increase in GAP43 and MARCKS proteinphosphorylation (Fukura et al., 1996). These results suggest thatthe ability of GABA to raise intracellular Ca21 levels may becentral to its functional role during development.

The cAMP signal transduction pathway is a potent regulator ofsynaptic neural physiology (for review, see Anholt, 1994; Cooperet al., 1994). A primary mechanism by which cAMP regulatesneural excitability is through the activation of cAMP-dependent

Received Nov. 1, 1996; revised March 19, 1997; accepted April 3, 1997.This work was supported by National Institutes of Health Grants NS10174 and

NS34887, the National Science Foundation, and Air Force Office of ScientificResearch.

Correspondence should be addressed to Anthony N. van den Pol, Department ofNeurosurgery, Yale University, School of Medicine, 333 Cedar Street, New Haven,CT 06520.Copyright © 1997 Society for Neuroscience 0270-6474/97/174785-15$05.00/0

The Journal of Neuroscience, June 15, 1997, 17(12):4785–4799

protein kinase A (PKA). The neuromodulatory actions of PKAhave been documented extensively and include altering ion chan-nel activity (Nagel et al., 1992; Johnson et al., 1994; Surmeier etal., 1995), gene expression (Impey et al., 1996), and neurotrans-mitter release (Sciancalepore and Cherubini, 1995; Huang et al.,1996). Although a considerable amount of information is knownabout the effects of PKA at the molecular and single channellevels, much remains to be determined about how its actions at avariety of sites may affect cytosolic Ca21 regulation in synapticallyactive neurons. For example, PKA has been shown to reduce theamplitude of the GABAA receptor conductance (Moss et al.,1992), to decrease voltage-gated Na1 channel currents (Smith andGoldin, 1996), and to potentiate kainate-induced currents (Guand Moss, 1996). A question of particular interest is howGABAergic activity mediating Ca21 elevations may be regulatedduring early development by PKA.

Similar to GABA, PKA has also been shown to play an impor-tant role during development. The cAMP signal transductionpathway has been implicated in a variety of developmentallyregulated processes, including cell cycle regulation (Grieco et al.,1996), neural migration (Behar et al., 1995), and gene induction(Zhang et al., 1993). Additionally, many neurotransmitters andneuropeptides that affect neural physiology through the regula-tion of adenylyl cyclase activity, including neuropeptide Y (NPY),dopamine, glutamate, and serotonin, are expressed in early devel-opment and could serve to modulate the Ca21-elevating actions ofGABA (Rajaofetra, 1989; Belin et al., 1991; Marti et al., 1992; vanden Pol et al., 1995, 1996a; Lieb et al., 1996). Based on thesefindings, we studied the modulation of GABAergic Ca21 elevatingactivity by the cAMP-mediated signal transduction pathway.

Here we report that Ca21 rises regulated by synaptic GABArelease during early development are dramatically influenced byactivation or inhibition of the cAMP-dependent signal transduc-tion cascade.

MATERIALS AND METHODSTissue culture. The mediobasal hypothalamus was removed from embry-onic day 18 Sprague Dawley rats. The tissue was enzymatically digested ina mild protease solution (10 U/ml papain and 0.2 mg/ml L-cysteine inEarl’s balanced salt solution) for 30 min. Next, the tissue was pelleted,and the protease solution was removed. Tissue was then suspended instandard tissue culture medium (glutamate- and glutamine-free DMEMsupplemented with 10% fetal bovine serum, 100 U/ml penicillin/strepto-mycin, and 6 gm/l glucose) and then triturated into a single-cell suspen-sion. Cells were washed and pelleted an additional three times. Thesingle-cell suspension was plated onto 22 mm2 glass coverslips that hadbeen coated with high-molecular-weight (540,000 Da) poly-D-lysine.High-density cultures (200,000/cm2) were used for all experiments. Hy-pothalamic neural cultures were maintained in a Napco 3600 incubator(37°C and 5% CO2) until they were ready for use. To limit non-neuronalcell proliferation, cytosine arabinofuranoside (1 mM) was added to thetissue culture medium 1 d after plating.

Fura-2 and fluo-3 Ca21 digital imaging. Cells were loaded for 20 minwith either 5 mM fura-2 AM or fluo-3 AM in standard perfusion solution(137 mM NaCl, 25 mM glucose, 10 mM HEPES, 5 mM KCl, 1 mM MgCl2,3 mM CaCl2, pH 7.4). The cells were then washed and allowed to recoverfor 15 min before the start of the experiment. Coverslips were thenloaded into a laminar style perfusion chamber. Solutions rapidly movedas a straight wave through the perfusion chamber, and complete washoutof the chamber occurred in ;5 sec. For fura-2, neurons were imagedusing a 403 Olympus objective with high 340/380 nm transmittance on aNikon Diaphot 300 inverted microscope. Fluo-3 experiments were per-formed using a 1003 Olympus objective. Unless noted otherwise, Ca21

digital recordings were made from the cell soma. All experiments wereperformed at room temperature.

A 486 PC clone was used to collect data, run Ca21 analysis software(Fluor; Universal Imaging Corporation, West Chester, PA), and control

the Lambda-10-filtered wheel driver (Sutter Instruments). Sixteen (500msec) digital frames of data were collected every 3 sec. Excitation lightcame from a 150 W Xenon lamp. The equation [Ca21]i 5 Kd(R 2Rmin)/(Rmax 2 R) was used to convert fura-2 ratiometric fluorescent Ca21

values to free Ca21 concentrations. R is the ratio of the two fluorescenceintensities, Rmin is the ratio in the absence of Ca21, and Rmax is the ratioin a saturating concentration of Ca21. The Kd for binding of Ca21 to fura2 was taken to be 224 nM (Grynkiewicz et al., 1985). Data for fluo-3fluorescence are represented on a 0–255 U scale. As standard protocolfor both fura-2 and fluo-3 imaging, background fluorescence values weresubtracted.

To determine the effects of different receptor agonists or signal trans-duction modulators on Ca21 rises triggered by the synaptic release ofGABA, the mean Ca21 rise from the Ca21 level in the presence of theGABAA receptor antagonist bicuculline was determined over a 15 secperiod just before the application of a receptor agonist or signal trans-duction modulator. The mean Ca21 rise was then determined over a 15sec period 90 sec after administration of the receptor agonist or signaltransduction modulator. Data for Ca21 rises for the two conditions arereported as a mean (pooled) Ca21 rise of all responsive neurons 6 SEM.Thirty to 50% of the neurons exhibited a Ca21 rise on bicucullineremoval. For assays that evoked a Ca21 response, either through agonistadministration to the perfusion solution or through electrical stimulation,the maximal Ca21 rise for individual neurons was determined by sub-tracting the mean basal Ca21 level for a 15 sec period just beforestimulation from the peak-evoked Ca21 rise. Ca21 responses from a totalof 1165 neurons were recorded in the course of these experiments.Modulation of GABA-related activity could refer to either a presynaptic orpostsynaptic site of action, whereas modulation of GABA-evoked Ca21

rises refers to a postsynaptic site of action.Electrical stimulation. Electrodes from a Grass SD9 stimulator were

placed at both ends of the perfusion chamber. Ca21 rises were stimulatedby passing 2–5 V/cm2, at 20 Hz frequency and 2 msec duration, throughthe chamber for 6 sec. Ca21 rises were visible immediately and usuallypeaked 4 sec after current application. At these voltages, Ca21 rises wereinhibited by either the application of the voltage-dependent Na1 channelblocker tetrodotoxin (TTX; 1 mM) or the administration of fast excitatoryneurotransmitter antagonists. At voltages higher than those used in thispaper (.6 V/cm2), TTX did not block the Ca21 response, suggesting anadditional nonsynaptic mechanism for the induction of Ca21 rises.

Immunostaining. Neurons were fixed for 1 hr in cold (220°C) methanoland then treated for 30 min with 3% BSA and 0.1% Triton X-100 in PBS.Neurons were then washed and incubated with mouse anti-synapsin 1antibody (1:100) (Chemicon, Temecula, CA). The synapsin antiserumrecognized a band of the correct weight on Western blots, and preab-sorption with synapsin antigen blocked staining (Smith et al., 1994). Ratanti-a-tubulin antibody (Sera Lab) was used at 1:200 for 30 min. Neuronswere washed and incubated with FITC- and Texas Red-labeled secondaryantibodies (Jackson Laboratory, Bar Harbor, ME) for 30 min. Afterwashing, cell fluorescence was visualized with the appropriate filter sets.In the fluorescent microscope, synapsin-immunostained boutons weregreen, and tubulin immunoreactivity was red.

Reagents. SKF-38393, McN-A-343, cytosine arabinofuranoside, GABA,cAMP, 8-bromo-cAMP (8-Br-cAMP), 8-(4-chlorophenylthio)-cAMP (4-Cl-cAMP), and poly-D-lysine were acquired from Sigma (St. Louis, MO).SQ-22,536, forskolin, (6)22-amino-5-phosphonopentanoic acid (AP5),6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), bicuculline, nimodipine,and TTX were acquired from Research Biochemicals (Natick, MA). NPYwas acquired from Peninsula Labs. Papain was acquired from Worthing-ton (Freehold, NJ); DMEM was from Life Technologies (Gaithersburg,MD); and fura-2 AM and fluo-3 AM were from Molecular Probes(Eugene, OR).

RESULTScAMP signal transduction pathway activationTo address the possibility that activating the cAMP signal trans-duction pathway affects GABA activity that mediates Ca21 ele-vations, membrane-permeable cAMP analogs that mimic the cel-lular effects of cAMP were administered to synaptically activehypothalamic neurons. Figure 1A shows that the withdrawal ofthe GABAA receptor antagonist bicuculline (20 mM) from theperfusion solution elicited a rapid and stable Ca21 rise, indicatingthat cytosolic Ca21 was raised by synaptically released GABA.

4786 J. Neurosci., June 15, 1997, 17(12):4785–4799 Obrietan and van den Pol • cAMP Modulates GABA-Evoked Ca21 Rises

Addition of 8-Br-cAMP (1 mM) caused a rapid reduction in Ca21

levels. The mean 6 SEM Ca21 rise after removal of bicucullinewas 73 6 6 nM. After the addition of 8-Br-cAMP the Ca21 risedecreased to 28 6 5 nM, representing a statistically significant( p , 0.0001, two-tailed t test) reduction in GABA-mediated Ca21

rise of 62% (n 5 68). Figure 1B shows that mean inhibition ofGABA activity regulating Ca21 rises by 8-Br-cAMP was dose-dependent. GABA-mediated Ca21 levels were increased in someneurons, but only at low 8-Br-cAMP concentrations. Specifically,the Ca21 level in 6 of 20 neurons was raised by 50 mM extracellular8-Br-cAMP, and at 150 mM 8-Br-cAMP, the Ca21 level was raisedin 4 of 30 neurons. However, at higher concentrations (500 mM

and 1 and 5 mM) 8-Br-cAMP only depressed Ca21 levels and didnot elevate Ca21 levels in any neurons (n 5 90).

Another membrane-permeable cAMP analog, 4-Cl-cAMP (2mM), also triggered a rapid Ca21 depression (Fig. 2A). Theeffectiveness of 4-Cl-cAMP was also dose-dependent; at 2 mM,4-Cl-cAMP caused a statistically significant ( p , 0.0001, two-tailed t test) 81% Ca21 depression (n 5 29), whereas 200 mM

4-Cl-cAMP reduced the GABA-mediated Ca21 rise by a statisti-cally significant ( p , 0.001, two-tailed t test) 39% (n 5 25). Figure2B shows that the coadministration of the potent PKA inhibitorRp-39,59-cyclic monophosphothioate triethylamine (Rp-cAMPS)largely blocked the effects of 4-Cl-cAMP, suggesting that theactions of membrane-permeable cAMP analogs are the result ofPKA activation. Application of 4-Cl-cAMP (1 mM) triggered a78% decrease in GABA-mediated Ca21 levels, whereas 4-Cl-cAMP (1 mM) administration, in the presence of Rp-cAMPS (200

Figure 1. Membrane-permeable analogsof cAMP trigger reversible Ca21 depres-sion. A, The removal of bicuculline (BIC,20 mM) from the perfusion solution trig-gered a rapid and sustained Ca21 rise inthe two representative neurons. Repeatedapplication of 8-Br-cAMP (1 mM) induceda rapid depression of the GABA-relatedCa21 rises that lasted only as long as 8-Br-cAMP was applied. B, Dose–response ef-fect of 8-Br-cAMP on the synaptically re-leased GABA-mediated Ca21 level. TheCa21 rise from a bicuculline-defined base-line was determined over a 15 sec periodjust before and 90 sec after 8-Br-cAMPapplication. The white bar (set at 100%) isthe normalized Ca21 rise just before 8-Br-cAMP application. Error bars indicateSEM. N, Total number of neurons as-sayed. C, 8-Br-cAMP had no effect onneuronal Ca21 levels if synaptic GABArelease was inhibited by the administra-tion of the Na1 channel blocker TTX (1mM). Neurons with only small baselineCa21 fluctuations were shown, so that anyeffect of 8-Br-cAMP on basal Ca21 mightbe observed. D, Membrane-impermeablecAMP (1 mM) had little extracellular ef-fect on GABA-mediated Ca21 levels. Allexperiments were performed in the pres-ence of AP5 (100 mM) and CNQX (10mM). All synaptic release experimentswere performed with embryonic day 18neurons after 5 DIV. In A, C, D, the baralong the x-axis shows time in min, and thebar to the left of each Ca21 trace is thecalibrated cytosolic Ca21 value for eachneuron.

Obrietan and van den Pol • cAMP Modulates GABA-Evoked Ca21 Rises J. Neurosci., June 15, 1997, 17(12):4785–4799 4787

mM), resulted in a 46% Ca21 decrease (n 5 42). As with lowconcentrations of 8-Br-cAMP, the administration of relatively lowlevels of 4-Cl-cAMP triggered Ca21 rises that were not observedat higher concentrations. Figure 2C shows two neurons treated toboth low (20 mM) and high (1 mM) concentrations of 4-Cl-cAMP.4-Cl-cAMP at a concentration of 20 mM triggered an enhancementin both the mean Ca21 concentration (Ca21 concentrations shownfor each bracketed region 6 SEM) and, in some neurons, the sizeof GABA-mediated Ca21 transients. By comparing the meanCa21 rise during bracketed region B (after bicuculline with-drawal) with the mean Ca21 rise during bracketed region C(during 20 mM 4-Cl-cAMP), we found that 4-Cl-cAMP triggered a.20% increase in GABA-mediated Ca21 rises in 17 of 57 neu-

rons. This effect is in striking contrast to the large Ca21 decreaseobserved on administration of 1 mM 4-Cl-cAMP. These neuronswere cultured on embryonic day 18 and maintained for 5 d in vitro(DIV). Figures often show data records from two or more neu-rons recorded simultaneously. This serves both to validate subtle,but consistent, effects of pharmacological manipulations and toshow the heterogeneity of neural response characteristics.

To remove any complicating effects of synaptically releasedglutamate on Ca21 rises, neurons were constantly perfused withthe ionotropic glutamate receptor antagonists AP5 (100 mM) andCNQX (10 mM). In the presence of glutamate receptor blockersAP5 and CNQX, basal Ca21 levels did not change when neuronswere switched between perfusion solutions containing bicuculline

Figure 2. Modulation of GABA activitymediating Ca21 rises. A, The membrane-permeable analog of cAMP, 4-Cl-cAMP (2mM), elicited a rapid and reproducible Ca21

depression. B, Coadministration of the pro-tein kinase A inhibitor Rp-cAMPS (200 mM)largely blocked the Ca21 depression elicitedby 4-Cl-cAMP (1 mM). C, Two representa-tive neurons show that administration of alow concentration of 4-Cl-cAMP (20 mM)triggered a Ca21 increase, whereas a highconcentration of 4-Cl-cAMP (1 mM) trig-gered a Ca21 depression. Numbers undereach bracket refer to the mean 6 SEM Ca21

concentration for the period within thebracketed region. Twenty time points (3 secinterval) were used to determine each Ca21

concentration, except for the beginning ofthe experiment [bicuculline (BIC) adminis-tration], at which 10 time points were used.Letters under brackets identify each brack-eted region.


(20 mM) or the Na1 channel blocker TTX (1 mM), suggesting thatin the absence of glutamatergic neurotransmission, GABA wasthe sole transmitter responsible for increasing neuronal Ca21

levels (data not shown).The Na1 channel blocker TTX (1 mM) inhibited Ca21 tran-

sients mediated by the synaptic release of GABA (Fig. 1C).8-Br-cAMP (1 mM) had no independent effect on basal Ca21

levels (Fig. 1C). As an additional control we added cAMP (1mM) to the perfusion solution. Because cAMP is membrane-impermeable, it should not mimic the intracellular effects of8-Br-cAMP or 4-Cl-cAMP. Figure 1 D shows that cAMP hadvery little effect on GABA-mediated Ca21 rises, suggesting that8-Br-cAMP and 4-Cl-cAMP were acting primarily through anintracellular mechanism to depress Ca21 levels rather than atan extracellular receptor. The Ca21 rise before the addition ofcAMP was 101 6 10 nM; during cAMP application, the Ca21

rise decreased to 87 6 10 nM, representing a statisticallyinsignificant ( p . 0.05, two-tailed t test) 14% cAMP-dependent decrease (n 5 38).

8-Br-cAMP inhibits GABA activity through presynapticand postsynaptic mechanismsTo determine whether activation of the cAMP-dependent signaltransduction cascade inhibits GABA-related Ca21 rises through apresynaptic or postsynaptic mechanism, we compared the effectsof 8-Br-cAMP on postsynaptic Ca21 rises elicited by bath appli-cation of GABA with the effects of 8-Br-cAMP on electricallystimulated Ca21 rises dependent on presynaptic GABA release.Figure 3A shows Ca21 rises elicited by bath application of GABA(10 mM). Interestingly, Ca21 rises were either inhibited (top trace)or potentiated (bottom trace) by 8-Br-cAMP (1 mM). Relative tothe control Ca21 rise immediately preceding 8-Br-cAMP admin-istration, 8-Br-cAMP depressed the Ca21 rise by .20% in 32 of109 neurons and potentiated the GABA-evoked Ca21 rises in 19of 109 neurons by .20%. In the absence of 8-Br-cAMP, repeatedpeak Ca21 rises evoked by GABA did not vary by .20% in any ofthe 109 neurons assayed. Figure 3C is a scatter plot analysis of themodulatory effects of 8-Br-cAMP administration on GABA-evoked Ca21 rises. Each point (neuron) is a representation of thefirst Ca21 rise in the presence of 8-Br-cAMP divided by thecontrol Ca21 rise immediately preceding 8-Br-cAMP administra-tion for each of the 109 neurons assayed. Data are displayed aspercentages. Zero percent on the y-axis signifies that the peak risein the presence of 8-Br-cAMP was equivalent to the control peakCa21 rise (no modulation); values ,0% represent depression, andvalues .0% represent response potentiation. Of note is the dualeffects (both potentiation and depression) of 8-Br-cAMP onGABA-evoked Ca21 rises. To ensure that the GABA-evokedCa21 rise was purely postsynaptic in nature, the neurons werecontinuously perfused with TTX (1 mM) to block action potential-dependent synaptic release of neurotransmitters.

To determine that GABA was released by an action potential-dependent mechanism at this early stage of development, a seriesof experiments was performed on neurons at 4 DIV. Electricalstimulation caused a large Ca21 rise in both neural cell bodies andneurites. Neurites loaded with fluo-3 (Fig. 4A) show a largeelectrically evoked Ca21 rise localized to small regions, suggestingdendrite segments postsynaptic to axonal boutons (Fig. 4D). ThisCa21 rise was reduced by bath application of either TTX (Fig. 4B)or bicuculline (Fig. 4C). These results suggest that actionpotential-mediated presynaptic release of GABA was responsiblefor the Ca21 rise. Ca21 rises occurred in the presence of the

glutamate receptor antagonists AP5 (100 mM) and CNQX (10mM), ruling out synaptic glutamate release as the cause of theCa21 rise. To demonstrate further that synapses were formed atthis developmental stage, cells were immunostained with synapsinantiserum. Highly localized, punctate synapsin staining is shown inFigure 4E. For control purposes, these cells were also immuno-stained for the structural protein a-tubulin (Fig. 4F). In contrastto the punctate synapsin staining, tubulin immunoreactivity wasfound throughout neurons and glia.

Figure 3B shows that electrical stimulation triggered reproduc-ible Ca21 rises in the neural cell soma. The addition of either TTX(1 mM) or bicuculline (20 mM) to the perfusion solution blockedthe rise. As in neurites, Ca21 rises occurred in the presence of theglutamate receptor antagonists AP5 (100 mM) and CNQX (10mM). Under this condition, the addition of 8-Br-cAMP (1 mM)caused a large depression in the electrically evoked Ca21 rise.Relative to the control electrically stimulated Ca21 rise immedi-ately preceding 8-Br-cAMP administration, 8-Br-cAMP de-pressed the Ca21 rise by .20% in 40 of 49 neurons. No neuronsexhibited a .20% potentiation of the peak evoked Ca21 re-sponse. A scatter plot analysis of the effects of 8-Br-cAMP onelectrically evoked Ca21 rises in 49 neurons is shown in Figure 3D.Of note is the largely inhibitory actions of 8-Br-cAMP on electri-cally evoked Ca21 rises. This is in contrast with data for purelypostsynaptically evoked Ca21 rises (Fig. 3C), suggesting that 8-Br-cAMP also affects GABA activity mediating Ca21 rises throughpresynaptic regulation of GABA release.

Figure 3E compares the modulatory effects of 8-Br-cAMP onCa21 rises elicited by bath-applied GABA with electricallyevoked release of presynaptic GABA. For a comparison ofeffectiveness of 8-Br-cAMP on postsynaptic (GABA-evoked)and presynaptic plus postsynaptic (electrically induced GABArelease) responses, data for Figure 3E were collected fromneurons that exhibited a peak Ca21 rise from 120 to 140 nM

during the second (control) GABA-evoked Ca21 rise. Ca21

rises of approximately equivalent levels were used so that apresynaptic component of 8-Br-cAMP-mediated Ca21 modu-lation could be fairly subtracted from the purely postsynapticmodulation of GABA-evoked Ca21 rises. Of interest was thefinding that the mean Ca21 rise evoked by bath application ofGABA was only slightly depressed (13%; statistically insignif-icant, P . 0.05, two-tailed t test) by 8-Br-cAMP, whereaselectrically induced Ca21 rises dependent on synaptic GABArelease were highly significantly ( p , 0.0001, two-tailed t test)reduced (61%). This difference in 8-Br-cAMP depression ofelectrically versus GABA-evoked Ca21 rises (48%) was prob-ably the result of inhibited GABA release. Unlike the otherexperiments, which used 5 DIV cultures, 4 DIV cultures wereused for electrical stimulation experiments. At this time pointduring development, neurons were synaptically connected,yet the Ca21-elevating action mediated by the spontaneousrelease of GABA was relatively low. Taken together, thesedata suggest that cAMP-dependent processes regulate Ca21

rises by decreasing the amount of presynaptic GABA releaseand to a lesser extent by altering postsynaptic Ca21 responsive-ness to GABA.

Modulation of adenylyl cyclase activityTo determine whether altering endogenous cAMP levels wouldmodulate GABA synaptic activity mediating Ca21 rises, neuronswere treated either with forskolin (an adenylyl cyclase activator)to increase cAMP levels or with SQ-22,536 (an adenylyl cyclase


Figure 3. 8-Br-cAMP reduces presynaptic re-lease of GABA. A, Brief (15 sec) repeated exog-enous administrations of GABA (10 mM) (ar-rows) to the perfusion solution elicited rapidand reproducible postsynaptic Ca21 rises. 8-Br-cAMP (1 mM) coadministration was capable ofeither enhancing or depressing the amplitude ofGABA-evoked Ca21 rises (see text). To ensurethat GABA-evoked Ca21 rises were exclusivelypostsynaptic in nature, the neurons were contin-uously perfused with TTX (1 mM) to block actionpotential-dependent presynaptic transmitter re-lease. B, Repeated electrical stimulation (E.S.)triggered reproducible Ca21 rises triggered bypresynaptic GABA release. Administration ofTTX (1 mM) or bicuculline (BIC, 20 mM) blockedthe Ca21 rise, suggesting that electrical stimula-tion triggered action potential-dependent synap-tic release of GABA. Administration of 8-Br-cAMP (1 mM) largely blocked the GABAactivity. This experiment was performed in theconstant presence of AP5 (100 mM) and CNQX(10 mM) to block the Ca21 rise elicited by gluta-mate receptor activation. C, D, Analysis of themodulatory effects of 8-Br-cAMP administrationon GABA-evoked Ca21 rises (C) and on electri-cally evoked Ca21 rises (D). Circles represent thefirst Ca21 rise in the presence of 8-Br-cAMPdivided by the control Ca21 rise immediatelypreceding 8-Br-cAMP administration; each circlerepresents the response of a single neuron. Re-sults are displayed as percentages of the controlCa21 rise. The 0% point on the y-axis signifiesthat the rise in the presence of 8-Br-cAMP wasequivalent to the control Ca21 rise. Values .0%represent response potentiation; values ,0%represent depression. E, The effects of 8-Br-cAMP on bath application of GABA and elec-trically stimulated synaptic release of GABA onCa21 rises were compared. Only neurons withcontrol evoked Ca21 rises (second Ca21 rise, justbefore 8-Br-cAMP) from 120 to 140 nM wereused to allow for an equitable comparison. SeeResults for details. Error bars indicate SEM. Themean GABA-evoked Ca21 rise was slightly de-pressed by the administration of 8-Br-cAMP(postsynaptic effect), whereas the electricallyevoked rise was dramatically depressed by 8-Br-cAMP administration (presynaptic plus postsyn-aptic). The electrically evoked Ca21 rise wasvirtually abolished by TTX or bicuculline. Elec-trical stimulation experiments were performedafter 4 DIV, a period when synaptic connectionsare found in most neurons.


inhibitor) to decrease cAMP levels. Figure 5A shows two repre-sentative neurons exhibiting four Ca21 rises: two Ca21 risesbefore and two Ca21 rises after a 15 min application of forskolin(20 mM). The administration of forskolin greatly depressed(.70%) the Ca21 rises elicited by bicuculline withdrawal. Theeffects of forskolin on GABA-mediated Ca21 rises are quantifiedin Figure 5B; 1st and 2nd refer to the two Ca21 rises elicited bybicuculline withdrawal before forskolin administration, whereas

3rd and 4th refer to the Ca21 rises after forskolin administration.A comparison of the first two with the second two Ca21 risesshows that the effects of forskolin were statistically significant( p , 0.0001, two-tailed t test).

Interestingly, SQ-22,536 (100 mM) also depressed the Ca21 riseelicited by bicuculline withdrawal (Fig. 5C). As with forskolin, a 15min pretreatment caused a statistically significant ( p , 0.05,two-tailed t test) decrease (.29%) in the mean Ca21 rise initiated

Figure 4. Electrical stimulation triggersGABA-mediated Ca21 rises in neurites. A,Fluo-3-loaded neurites are shown undercontrol conditions (no electrical stimula-tion). B, In the presence of TTX (1 mM),electrical stimulation does not elicit a Ca21

rise. C, Electrically induced Ca21 rise is alsolargely inhibited by bath application of bicu-culline (20 mM). D, In the absence of bicu-culline and TTX, large and localized (redarrows) Ca21 rises were triggered by elec-trical stimulation. These experiments wereperformed in the presence of AP5 (100 mM)and CNQX (10 mM). A9–D9, High magnifi-cation of areas from A–D shown by the boxin A. The color bar shows color codes of lowand high Ca21 levels. Scale bar, 2.5 mm. E,Neurons immunostained for synapsin I.Blue arrows indicate punctate staining, cor-responding to synapse location. F, Sameregion immunostained for a tubulin. Yellowarrows identify the same neurite in bothmicrographs. Scale bar, 8 mm.


by bicuculline withdrawal (Fig. 5D). Together, these results indi-cate that both increasing and decreasing cAMP levels throughmodulating adenylyl cyclase activity reduces GABA-mediatedCa21 rises.

Protein kinase A inhibitionTo determine whether a basal level of PKA-mediated phos-phorylation plays a role in maintaining the level of GABA Ca21

rises, we assessed the effect of the potent PKA inhibitors H89and Rp-cAMPS. Figure 6 A shows that the administration ofRp-cAMPS (200 mM) to synaptically active neurons initiated arapid Ca21 depression. The removal of bicuculline caused amean Ca21 rise of 70 6 6 nM. Addition of Rp-cAMPS de-creased the Ca21 rise to 18 6 2 nM, representing a statisticallysignificant ( p , 0.0001, two-tailed t test) 74% decrease in Ca21

activity (n 5 45). Of interest was the finding that the Ca21

depression triggered by Rp-cAMPS persisted for an extendedperiod even after the Rp-cAMPS was washed out. Fifty-eightpercent of the neurons assayed (26 of 45) did not recover.50% of their pre-Rp-cAMPS Ca21 level during any time afterRp-cAMPS withdrawal. In control experiments, none of 35unstimulated neurons showed .50% reduction in GABA Ca21

rises over an identical period.Rp-cAMPS also inhibited the induction of Ca21 rises mediated

by the synaptic release of GABA (Fig. 6B). The two representa-tive neurons in Figure 6B initially exhibited three robust Ca21

rises, whereas after Rp-cAMPS pretreatment, bicuculline with-drawal did not initiate a Ca21 rise. As did Rp-cAMPS, a short, 2min application of H89 (15 mM) triggered a rapid Ca21 depression(Fig. 6C). H89 reduced the GABA-mediated Ca21 rise from 44 65 to 12 6 1 nM (n 5 14). The level of H89-mediated depression

Figure 5. Adenylyl cyclase modulationalters GABA Ca21 rises. A, Before theaddition of adenylyl cyclase modulators,withdrawal of bicuculline (BIC, 20 mM)elicited rapid, reproducible Ca21 re-sponses. A, After a 15 min administra-tion of the adenylyl cyclase activator for-skolin (20 mM), GABA-mediated Ca21

rises elicited by the removal of bicucul-line from the perfusion solution weresignificantly depressed, relative to Ca21

rises elicited before forskolin administra-tion. B, Bar graph representation of themean Ca21 rises triggered by the fourbicuculline withdrawals; 1st and 2nd re-fer the Ca21 rises before forskolin ad-ministration, and 3rd and 4th refer to theCa21 rises after forskolin administration.C, Relative to the initial two Ca21 rises,the Ca21 rises elicited after the adminis-tration of the adenylyl cyclase inhibitorSQ-22,536 (100 mM) were significantlydepressed. The dashed line is meant toapproximate the mean GABA-mediatedCa21 rise before adenylyl cyclase modu-lators were added. D, Graphical repre-sentation of the mean Ca21 rises trig-gered by the four bicucullinewithdrawals; 1st and 2nd refer to the twoCa21 rises before SQ-22,536 administra-tion, and 3rd and 4th refer to the twoCa21 rises after SQ-22,536 administra-tion. Error bars indicate SEM. All exper-iments were performed in the presenceof AP5 (100 mM) and CNQX (10 mM).


was statistically significant ( p , 0.0001, two-tailed t test). As ameasure of the long-term effectiveness of H89, only 4 of the 14neurons assayed recovered .50% of their pre-H89 Ca21 level 30min after H89 withdrawal.

H89 also depressed GABA activity triggered by electrical stim-ulation. Figure 6D shows that the brief administration of H89 (15mM) resulted in a statistically significant ( p , 0.0001, two-tailed ttest) depression in the Ca21 rise. Electrical stimulation triggereda mean Ca21 rise of 441 6 26 nM. Addition of H89 reduced theCa21 rise to 170 6 12 nM (n 5 78). As in the endogenous activityassays described above, inhibition initiated by H89 persisted longafter it was washed from the perfusion chamber in some neurons.The addition of TTX or bicuculline largely blocked the electricallyevoked Ca21 rise.

PKA and voltage-dependent Ca21 channelsIn these experiments, we addressed the mechanism of postsynap-tic PKA actions. In the constant presence of TTX (1 mM), the bathapplication of GABA (10 mM) triggered a rapid Ca21 rise that wasdepressed by the H89 (15 mM) administration (Fig. 7A). After H89withdrawal from the perfusion solution, neuronal Ca21 respon-siveness to GABA slowly recovered toward pre-H89 levels. Be-cause GABA elicits a Ca21 rise through the activation of voltage-activated Ca21 channels (VACCs), the direct effect of H89 onhigh K1 (15 mM)-induced Ca21 rises triggered by VACC activa-tion was assessed. Similar to its effects on GABA, H89 adminis-tration rapidly depressed Ca21 rises mediated by VACCs (Fig.7B). After H89 withdrawal the recovery of Ca21 responsivenesshad a general appearance that was very similar to the recovery of

Figure 6. GABA Ca21 levels are reduced byprotein kinase A inhibitors. A, Administration ofthe protein kinase A inhibitor Rp-cAMPS (200mM) caused a rapid reduction in the Ca21 level.In the top neuron, the Ca21 level remained de-pressed for an extended period after the removalof Rp-cAMPS from the perfusion solution. B,Pretreating neurons with Rp-cAMPS (200 mM)blocked GABA-mediated Ca21 rise inductionelicited by bicuculline (BIC, 20 mM) removal. C,Another protein kinase A inhibitor, H89 (15mM), also rapidly depressed GABA-mediatedCa21 levels. As with Rp-cAMPS, the Ca21 de-pression triggered by H89 persisted for an ex-tended period after H89 withdrawal. D, The ad-ministration of H89 (15 mM) depressed Ca21

rises triggered by electrical stimulation (E.S., ar-rows) of GABA release. Electrically inducedCa21 rises could be blocked by tetrodotoxin orbicuculline (not shown here). As with its effectson spontaneous GABA release, the effects ofH89 persisted even after it was withdrawn fromthe perfusion solution.


Figure 7. PKA inhibition reduces evokedCa21 responsiveness. A, GABA (10 mM)(arrows) elicited rapid Ca21 rises that weredepressed by the administration of thePKA inhibitor H89 (15 mM) in two neu-rons. B, H89 also depressed Ca21 rises ini-tiated by high K1 (15 mM) (arrows) admin-istration. C, D, Analysis of the modulatoryeffects of H89 on GABA-evoked Ca21 rises(C) or high K1-evoked Ca21 rises (D). Cir-cles are percentage representations of thefirst Ca21 rise in the presence of H89 di-vided by the control Ca21 rise immediatelypreceding H89 administration; each circlerepresents the response of a single neuron.The 0% point on the y-axis signifies that theevoked rise in the presence of H89 wasequivalent to the evoked Ca21 rise. Values.0% represent potentiation; values ,0%represent depression. E, The L-type Ca21

channel blocker nimodipine (1 mM) largelyblocked Ca21 rises elicited by high K1.Both GABA and high K1 were applied for15 sec. F, Graphical representation of themean Ca21 rises elicited either by GABAor high K1 before (white bars) or duringthe coadministration of H89 (black bars) ornimodipine (striped bar). N, Total numberof neurons assayed. Error bars indicateSEM.


GABA responsiveness, suggesting that the H89-mediated depres-sion of GABA responses may result, in part, from VACC inhibi-tion. Figure 7, C and D, shows scatter plot analyses of the effectsof H89 on GABA-evoked (n 5 105) or high K1-evoked (n 5 104)Ca21 rises, respectively. Single-cell values were determined bydividing the first evoked Ca21 rise in the presence of H89 by thecontrol-evoked Ca21 rise immediately preceding H89 administra-tion. Values are expressed as percentages of the control Ca21 rise.A Ca21 rise in the presence of H89 that was larger than controlrises was .0%, whereas a rise of smaller peak height than thecontrol rise was ,0%; rises of equal peak height were 0%. Ofnote, the overall level of Ca21 depression elicited by H89 wasgreater for K1-evoked Ca21 rises than for GABA-evoked Ca21

rises. Figure 7E shows that high K1-induced Ca21 rises werelargely suppressed by the administration of the L-type Ca21

channel blocker nimodipine (1 mM). A graphical representation ofmean Ca21 rises in response to GABA and high K1 before andduring H89 or nimodipine application is shown in Figure 7F. Themean Ca21 depressions triggered by H89 and nimodipine werestatistically significant ( p , 0.0001, two-tailed t test). These find-ings suggest that tonic PKA-mediated Ca21 channel phosphory-lation is required for GABA to elicit a robust Ca21 rise.

Modulation of GABA Ca21 rises in adenylyl cyclase-coupled neurotransmitter receptor systemsThe experiments above used pharmacological tools to directlyalter different stages of intracellular pathways that affect kinase-mediated phosphorylation. We include the experiments below todemonstrate that activating neurotransmitter receptors that havepreviously been shown to modulate cAMP levels (either increaseor decrease) exert actions parallel to those occurring when agentsthat act downstream of the receptor are activated (i.e., experi-ments described above).

We tested whether transmitter receptors shown to be eitherpositively or negatively coupled to cAMP production alter GABACa21 rises. Toward this end, we chose the well characterizeddopamine D1 receptor. The D1 receptor has been shown to becoupled to an increase in cAMP levels (Shultz et al., 1987; Steffeyet al., 1991; Liu et al., 1992; Lovenberg et al., 1991). Administra-tion of the dopamine D1 receptor-specific agonist SKF-38393 (5mM) triggered a rapid and reproducible depression in the GABA-mediated Ca21 rise (Fig. 8A). The withdrawal of bicuculline fromthe perfusion solution triggered a mean Ca21 rise of 86 6 4 nM

from the basal Ca21 level in the presence of bicuculline. Additionof SKF-38393 caused the Ca21 rise to decrease to 56 6 3 nM,representing a statistically significant ( p , 0.0001, two-tailed ttest) 35% depression in the GABA-mediated Ca21 rise (n 5 79).Previously, we found that NPY (100 nM) caused a large (.70%)depression in the Ca21 rise elicited by synaptically releasedGABA (Obrietan and van den Pol, 1996b). An example of theCa21-depressing actions of NPY is shown in Figure 8B. Severalstudies have shown that NPY receptor stimulation triggers inhib-itory G-protein activation, leading to a decreased cAMP level(McAuley et al., 1991; Bleakman et al., 1992; Larhammar et al.,1992; Zhu et al., 1992). Additionally, administration of the mus-carinic acetylcholine receptor agonist McN-A-343 (100 mM) trig-gered a Ca21 depression (Fig. 8C). The withdrawal of bicucullinefrom the perfusion solution elicited a mean Ca21 rise of 96 6 7 nM

from the basal Ca21 level. McN-A-343 administration caused theCa21 rise to decrease to 65 6 5 nM, representing a statisticallysignificant ( p , 0.0001, two-tailed t test) 32% depression in theGABA-mediated Ca21 rise (n 5 52). A large number of studies

have shown that the muscarinic acetylcholine receptors modulatecAMP levels (McKinney et al., 1991; Schwarz et al., 1993; Burfordet al., 1995; Migeon et al., 1995). Interestingly, in 13 of 58neurons, a lower McN-A-343 concentration (15 mM) increasedGABA-mediated Ca21 levels by .10%. In contrast, only 1 of 52

Figure 8. Ca21 rises mediated by GABA activity are depressed by selec-tive activation of neurotransmitter receptors coupled to adenylyl cyclaseregulation. The removal of bicuculline (BIC, 20 mM) from the perfusionsolution triggered a rapid and sustained Ca21 rise in the three represen-tative neurons. A, Stimulation of the D1 receptor by the administration ofSKF-38393 (5 mM) reduced the amplitude of the spontaneous GABA-mediated Ca21 rise. B, NPY (100 nM) triggered a rapid Ca21 depression.Blockade of the GABAA receptor with bicuculline at the end of theexperiment reduced Ca21 to basal levels. C, Stimulation with the musca-rinic acetylcholine receptor agonist McN-A-343 (100 mM) also triggeredCa21 depression. The ionotropic glutamate receptor antagonists AP5 (100mM) and CNQX (10 mM) were perfused throughout the experiment toblock synaptically released glutamate from triggering a Ca21 rise.


neurons treated with the higher concentration of McN-A-343 (100mM) showed a .10% increase in the GABA-mediated Ca21 rise.These results support the hypothesis that receptor systems cou-pled to either an increase or a decrease in cAMP production mayalter GABA Ca21 rises significantly.

DISCUSSIONActivation of the GABAA receptor, either through exogenousagonist application or synaptic GABA release, elicits a Ca21 risein the majority of developing neurons from many brain regions(Obrietan and van den Pol, 1995). In the present study, wecharacterized the functional role of the cAMP signal transductionpathway in terms of its ability to regulate GABA-related Ca21

rises. Our data suggest that either activating or inhibiting thecAMP signal transduction pathway significantly depressed theGABA activity detected with Ca21 imaging. The results of affect-ing the cAMP signal transduction pathway were similar to theeffects of stimulating transmitter receptors thought to be coupledto either a decrease or an increase in cAMP levels.

PKA activation or inhibition, as described in this paper, may causea variety of different mechanisms to work together or in opposition toone another, with the general effect being a decrease in the Ca21 riseinitiated by GABA-mediated neurotransmission. To our knowledge,ours is the first report characterizing the regulation of GABA-mediated Ca21 rises by direct modulation of the cAMP signal trans-duction pathway. These effects were observed with agents that eitheractivated (membrane-permeable cAMP analogs and forskolin) orinhibited (PKA inhibitors and SQ-22536) the cAMP signal transduc-tion pathway at several different points along the pathway.

Postsynaptic effectsBlocking PKA activity with H89 or Rp-cAMPS depressed peakCa21 rises elicited by the bath application of GABA. Because thepresynaptic release of GABA-containing vesicles was blockedwith TTX, the observed inhibitory effect of PKA was postsynapticin nature. A number of possible mechanisms could account for thedepression of GABA-mediated Ca21 levels observed when thecAMP signal transduction cascade was altered. A decrease inGABAA receptor activity resulting from an inhibition of steady-state GABAA receptor phosphorylation could explain the depres-sion resulting from PKA inhibition. Along these lines, severalstudies have shown that GABAA receptor-mediated activity isregulated by phosphorylation. GABAA currents were decreasedby .90% if cells were ATP-depleted (Chen et al., 1990), suggest-ing that a basal level of phosphorylation was required for main-tenance of optimal GABAA receptor channel function. In addi-tion, GABAA receptor conductance has been shown to be directlyaffected by PKA-mediated phosphorylation (Moss et al., 1992). Inthe hippocampus, the frequency of GABA-mediated giant depo-larizing potentials is modulated by 8-Br-cAMP or forskolin andinhibited by Rp-cAMPS (Strata et al., 1995). Within this context,our results suggest that tonic postsynaptic PKA-mediated phos-phorylation is required for optimal Ca21 responsiveness.

Ca21 responses triggered by the direct activation of VACCs(largely L-type) were also suppressed by H89. Because the abilityof GABA to elicit a Ca21 rise in hypothalamic neurons is depen-dent on L-type Ca21 channel activation (Obrietan and van denPol, 1995), these results suggest that H89-mediated depression ofGABA-evoked Ca21 rises may result, in part, from the inhibitionof L-type VACC activity. A basal level of phosphorylation may berequired for optimal L-type Ca21 channel activity; Ca21 currentsin HEK-293 cells transfected with the L-type Ca21 channel gene

were depressed by administration of PKA inhibitors (Perez-Reyeset al., 1994). Additionally, forskolin increased the L-type Ca21

current in ferret ventricular myocytes, and this increase wasblocked by H89 (Yuan and Bers, 1995). Our results in neurons areconsistent with these findings. This does not rule out a possibleadditional effect of phosphorylation on K1 channels that could, inturn, influence VACCs.

Presynaptic effectsThe cAMP signal transduction pathway appeared to act at bothpresynaptic and postsynaptic sites to affect GABA-related Ca21

rises. Ca21 rises triggered by electrical stimulation of presynapticGABA release were depressed to a much greater extent by 8-Br-cAMP than postsynaptic Ca21 rises elicited by the bath applica-tion of GABA. Additionally, postsynaptic Ca21 rises in a sub-population of neurons were potentiated by 8-Br-cAMP, whereaspotentiation was not observed when Ca21 rises were elicited byelectrically induced presynaptic GABA release. These differentialeffects suggest that the cAMP signal transduction pathway mayinhibit presynaptic GABA release, as well as modulate postsyn-aptic GABA responsiveness. This conclusion is consistent with theview that PKA has a spatially limited action and may exertindependent or opposing effects in presynaptic axons comparedwith the postsynaptic somatodendritic complex.

PKA activators 8-Br-cAMP or 4-Cl-cAMP decreased GABA-related Ca21 rises, although we also noted that low concentrationsof 8-Br-cAMP or 4-Cl-cAMP increased Ca21 levels in a subpopu-lation of neurons during endogenous activity assays. These Ca21

modulatory effects of 8-Br-cAMP and 4-Cl-cAMP were largelythrough a presynaptic mechanism. For a presynaptic receptorcoupled to adenylyl cyclase activation (which would result inincreased PKA activity), there have been a variety of reportsdescribing either potentiation or inhibition of transmitter release.For example, activation of the dopamine D1 receptor (coupled tocAMP production) facilitates neurotransmission in the hippocam-pus (Imperato et al., 1993) and in the ventral tegmetal area(Cameron and Williams, 1993), whereas D1 receptor stimulationhas been shown to decrease neurotransmission in the basal fore-brain (Momiyama et al., 1996) and in the shell region of thenucleus accumbens (Pennartz et al., 1992).

The exclusive effect of PKA inhibitors on Ca21 rises elicited bypresynaptic GABA release was inhibitory. Receptors coupled toadenylyl cyclase inhibition (which would result in decreased PKAactivity), including the GABAB receptor (Dittman and Regehr,1996), the adenosine A1 receptor (Potier and Dutar, 1993), andthe NPY receptor (Bleakman et al., 1992), have been shown todecrease transmitter release. These findings suggest that the ef-fects of PKA on transmitter release may depend on the presyn-aptic expression of a variety of phosphorylation targets that maybe differentially expressed in different brain regions, developmen-tal stages, or neural phenotypes. This is consistent with the pre-synaptic localization of many hypothalamic neuromodulatory re-ceptors that act through G-proteins to regulate cAMP (Chen andvan den Pol, 1996).

Long-term Ca21 depressionIn assays in which Ca21 rises were elicited via synaptic GABArelease, inhibition of PKA by H89 or Rp-cAMPS triggered arapid and long-term Ca21 depression (.30 min) in a largenumber of neurons (58%). Based on the differential postsyn-aptic effects of PKA inhibitors, long-term Ca21 depressionseems to be mediated through a largely presynaptic mecha-


nism. Interestingly, membrane-permeable cAMP analogs trig-gered short- but not long-term Ca21 depression also through alargely presynaptic mechanism. These results suggest that thecellular mechanisms that inhibit PKA-mediated phosphoryla-tion may be uniquely positioned to depress neural activity overextended periods.

NPY has both presynaptic and postsynaptic actions on GABAactivity in developing hypothalamic neurons. Whereas NPY trig-gered a predominately brief postsynaptic depression of GABA-evoked Ca21 rises (Obrietan and van den Pol, 1996b), presynapticactions of NPY on transmitter release were often long-term(Obrietan and van den Pol, 1996b; van den Pol et al., 1996c). Inaddition, NPY receptor stimulation depressed the Ca21 rise in asimilar percentage of neurons and for a similar duration as diddirect PKA inhibition. NPY has been shown both to decreasecAMP levels in neurons (Harfstrand et al., 1987; McAuley et al.,1991) and to act through a Gi/Go-protein-coupled mechanism toreduce Ca21 rises elicited by synaptic GABA release (Bleakmanet al., 1992; Obrietan and van den Pol, 1996b; van den Pol et al.,1996c). Activation of a Gi-protein-coupled mechanism could re-sult in decreased adenylyl cyclase activity and, in turn, decreasedPKA activity, an effect similar to adding Rp-cAMPS or H89 to theperfusion solution. These results indicate that the long-term ef-fects of NPY receptor activation on GABA-mediated Ca21 risesmay be the result of decreased PKA activity. Other receptorsystems coupled to adenylyl cyclase regulation have been shownto trigger extended depression of neural activity, includingthe GABAB receptor (Yang et al., 1994; Wagner and Alger,1995) and metabotropic glutamate receptor (Bolshakov andSiegelbaum, 1994; O’Mara et al., 1995). Immunohistochemicalanalysis has revealed a high level of adenylyl cyclase expressionboth in postsynaptic densities and in presynaptic axon terminals(Mons and Cooper, 1995), suggesting that adenylyl cyclase mayplay an important role both as a regulator of postsynaptic mem-brane ion conductance and presynaptic neurotransmitter release.

Biphasic effects of phosphorylation onGABA transmissionGABA-related Ca21 rises were depressed by both strong activa-tion and inhibition of the cAMP signal transduction system. Thisbiphasic response could be explained if the effects of PKA onGABA-mediated Ca21 rises were characterized by an invertedU-shape function (Fig. 9). During GABA neurotransmission, abasal level of tonic PKA-mediated phosphorylation results in anear maximal Ca21 level. This condition would put PKA activitynear the top of the inverted U-shape function. When PKA activityis radically altered, either through large increases (D1 receptoractivation, forskolin, and cAMP analogs) or decreases (NPYreceptor activation, SQ-22,536, Rp-cAMPS, and H89), PKA ac-tivity moves out of the region of the inverted U-shape functionthat provides maximal GABA-mediated Ca21 rises.

Low extracellular concentrations of 8-Br-cAMP (50–150 mM)or 4-Cl-cAMP (20 mM) increased GABA-mediated activity insome neurons, whereas high concentrations of 8-Br-cAMP ($500mM) or 4-Cl-cAMP ($200 mM) only decreased Ca21 levels. This isconsistent with the hypothesis that the basal level of PKA activityin some neurons is just below that needed to provide a maximallevel of GABA-mediated Ca21 rises. As described by an invertedU-shape response, this effect would result in a movement towardthe apex of the inverted U. The data fitted to this model indicatethat GABA-mediated Ca21 rises depend on ongoing PKA activ-

ity, and that PKA activity must be maintained within a narrowzone for GABA to elicit a maximal Ca21 rise.

Functional role of GABA-mediated Ca21 increasesduring early developmentChanges in cytosolic Ca21 levels affect a variety of developmen-tally regulated neural processes. Induction of Ca21 influx wasprimarily thought to be through the activation of classicmembrane-depolarizing transmitters such as glutamate. Recentdata have shown that GABA can exert a similar depolarizingaction in developing neurons. This Ca21-elevating action ofGABA is not restricted to hypothalamic neurons but was found inthe majority of developing neurons from eight brain regions,including hippocampus, spinal cord, cortex, olfactory bulb, andstriatum (Reichling et al., 1994; Obrietan and van den Pol, 1995),and after severe neural injury (van den Pol et al., 1996b). Theability of GABA to increase Ca21 levels suggests that it may havean important role during nervous system development. Alongthese lines, the GABA agonist muscimol can upregulate BDNFmRNA expression in rat hippocampal neurons, and this effect isblocked by the L-type Ca21 channel blocker nifedipine (Berningeret al., 1995). GABA secretion decreased DNA synthesis in corticalprogenitor cells (LoTurco et al., 1995). GABA induces motility ofembryonic cortical neurons through an increase in intracellularCa21 (Behar et al., 1996). Other GABA-mediated actions duringneural development include altering neurite outgrowth (Barbin etal., 1993), triggering chemokinesis (Behar et al., 1994), inducingGABA receptor expression (Meier et al., 1984), and regulatingneural phenotype (Marty et al., 1996). We have recently reported

Figure 9. Model of biphasic effect of PKA activity on GABA transmis-sion. Proposed model describing how increases or decreases in PKAactivity result in a reduction in GABA-mediated Ca21 rises. Arrows anddashed lines indicate the range of endogenous PKA activity required formaximal GABA-mediated Ca21 rises. See Discussion for details.


that GABA triggers localized Ca21 increases in developing neu-rites and growth cones (Obrietan and van den Pol, 1996a), sug-gesting a possible Ca21-dependent role for GABA in altering thedirectionality of growth cone motility and the rate of neuriteoutgrowth. Based on these reports and the findings in our paper,during early development the activation of neurotransmitter re-ceptors (such as NPY, glutamate, dopamine, serotonin, melato-nin, and acetylcholine) coupled to the cAMP signal transductionpathway could substantially alter GABA-related Ca21 rises. This,in turn, could affect the myriad of Ca21-dependent developmentalprocesses described above.

Depression of Ca21 rises attributable to an inhibitory action ofpresynaptic neuromodulators on GABA release is only foundduring early development. Because GABA does not raise Ca21 inolder neurons, but may instead decrease it (Obrietan and van denPol, 1995), neuromodulatory inhibition of GABA release in ma-ture neurons would be expected to exert the opposite effect onCa21. Thus, in developing neurons, modulatory inhibition ofGABA release reduces cytosolic Ca21 but in mature neuronswould raise Ca21.

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