was cultured on dextran beads in the presence of nerve growth factor for 7-10 days. Culture medium was formulated

Transcription

1 Pro. Nati. Aad. Si. USA Vol. 87, pp , Marh 1990 Neurobiology Habituation in the single ell: Diminished seretion of norepinephrine with repetitive depolarization of P12 ells (memory/protein phosphorylation/protein kinase /phorbol esters/aliwum) PHILIP N. MFADDN AND DANIL. KOSHLAND, JR. Department of Moleular and ell Biology, 401 Barker Hall, University of alifornia, Berkeley, A ontributed by Daniel. Koshland, Jr., Deember 4, 1989 ABSTRAT Neuronally differentiated P12 ells serete dereasing amounts of [3H]norepinephrine when repetitively stimulated by depolarizing onentrations of potassium ion. The dereasing response shows attributes that have been lassially asribed to response habituation, a behavior ommonly observed in nervous systems but found here in a homogeneous ell type. Alteration of the habituation pattern was aused by ativators of the protein kinase pathway and of voltage-gated alium hannels. Memory is defined as "the modifiation of behavior by experiene" (1) and is generally now onsidered to involve hanges indued at the ellular or synapti level of neurons. Several systems in whih stimuli alter subsequent behaviorlong-term potentiation (2) and habituation (3)-have been used as rudimentary models of memory, and more omplex systems (4) have been shown to have harateristis that overlap with these simpler systems. It would be of theoretial interest and pratial value if a single ultured ell system were shown to have these properties beause it would establish that feedbak from different types of ells is not neessary. Suh a system would allow the biohemistry of the proess to be studied more effetively beause many idential ells would be available. P12 ells, derived from a rat pheohromoytoma, have been widely used as a model neuronal system (5). When ultured with nerve growth fator, P12 ells develop long neuriti outgrowths (6), beome eletrially exitable (7), and take on many of the biohemial traits of sympatheti noradrenergi neurons (5). Membrane depolarization, mediated by raising the K+ onentration in the medium, evokes neuroseretion of ateholamines from these ells (8). a2+ influx through voltage-gated hannels is the proposed seond messenger for neuroseretion (9). Phorbol ester treatment inreases seretion by P12 ells (10), presumably through the ativation of protein kinase (11). To test whether neuronally differentiated P12 ells would be an appropriate model system for habituation and memory, ells were presented with repetitive membrane depolarization, and their neurotransmitter seretion was measured. The resulting ellular behavior satisfies several of the riteria that have been used to lassify habituation in organisms (12). The effets of a a2+-hannel agonist and protein kinase ativation by phorbol esters were also tested. The results reveal that habituation ours within single ells and is affeted by important seond messenger systems. XPRIMNTAL PRODURS ells and Media. The rat pheohromoytoma ell line P12 was obtained from the Amerian Type ulture olletion and The publiation osts of this artile were defrayed in part by page harge payment. This artile must therefore be hereby marked "advertisement" in aordane with 18 U.S solely to indiate this fat. was ultured on dextran beads in the presene of nerve growth fator for 7-10 days. ulture medium was formulated as RPMI 1640 growth medium/10% heat-inativated horse serum (56, 30 min)/5% fetal bovine serum/peniillin (20 units ml-1)/streptomyin (20 units ml-1)/20 mm sodium Hepes, ph 7.4 (6). Test media for seretion measurements were formulated as reported (8). Low K+ medium was 125 mm Nal/4.8 mm KI/1.3 mm al2/1.2 mm Mg S04/1.2 mm potassium phosphate/5.6 mm gluose/1 mm asorbate, and 25 mm Hepes, ph 7.4. High K+ medium was 69 mm Nal/56 mm KI and otherwise idential to low K+ test medium. Phorbol 12-myristate 13-aetate (PMA) (Sigma) and Bay K 8644 (albiohem) were added to media using dimethyl sulfoxide vehile at a final onentration of 0. 1% (vol/vol), a onentration of vehile that had no measurable effet on ellular seretion. [3H]Norepinephrine Loading of ells. ells (5 x 106) on dextran beads were suspended in 5 ml of low K+ medium to whih was added [3H]norepinephrine (L-[7,8-3H]norepinephrine; 20,i (1 i = 37 GBq) in 20,ul of vehile ontaining 20 mm aeti aid/ethanol (9:1); 30 i mmol-1 (Amersham). The suspensions were then inubated (370, laboratory atmosphere) for 1 hr with the radiolabel, with gentle swirling every 15 min. Approximately half of the radiolabel in the medium was taken up by the ells in this time and was evidently stored in seretory granules (13, 14) beause we found that >80% of the intraellular radiolabel would sediment after lysis of the ells with digitonin. The ell-overed beads (106 ells; 5 x 104 beads; 4 x 106 dpm) were then transferred to a perfused ell-hamber onstruted from a plasti-syringe barrel (0.15- ml internal volume) and with a glass-fiber filter at its outlet. [3H]Norepinephrine Seretion Measurements. ellular seretion of [3H]norepinephrine was measured by a method adapted from previous studies (15). Medium was delivered to the ell hamber by a peristalti pump (0.28 ml-min-1), the stream of medium exiting the ell hamber was mixed with a stream of sintillation fluid (2.2 ml-min-'), and the mixed streams then entered a flow-through sintillation detetor (Flo-One Beta; Radiomati Instruments and hemial, Tampa, FL). The number of pm of radioativity passing through the sintillation ounter was updated and reorded every 0.1 min. Seretion was evoked by swithing the medium oming into the ell hamber to high K+. The lag time between swithing to high K+ and the appearane of the response has been subtrated in all figures. [3H]Norepinephrine seretion measurements have been orreted for the ongoing loss of radiolabel and are displayed as the perentage of remaining ellular [3H]norepinephrine released per min. The total amount of radiolabel present in the ells was determined after lysis of the ells by detergent at the end of eah experiment with 1% (vol/vol) Triton X-100. During 1- to 3-hr experiments, between 10 and 30% of the initial ellular radiolabel was released, depending on the number of stimu- Abbreviation: PMA, 4,8-phorbol 12-myristate 13-aetate. 2031

2 2032 Neurobiology: MFadden and Koshland lations applied. Response sizes were measured by alulating the areas under the [3H]norepinephrine peaks after orretion had been made for the ongoing loss of radiolabel. RSULTS ellular Habituation with Repetitive Membrane Depolarization. The effet of repetitive stimulation with high K+ depolarization on norepinephrine seretion was studied in P12 ells. A single presentation of high K+ is known to stimulate norepinephrine seretion from P12 ells (8). The response of the ells, defined as the amount of [3H]norepinephrine sereted with eah high K+ stimulus, was found to diminish steadily with ertain patterns of repetitive stimulation, suh as the presentation of high K+ for 1 min every 5 min (Fig. 1). Other temporal patterns of stimulation were tested, and the derease in the response was found to depend on the frequeny of high K+ stimulation (Fig. 2). Habituation was strongest with frequent stimulation, suh as with 1-min stimulations every 5 or every 10 min, in whih the response dereased to -25% and 80%o of its initial size, respetively. The response size remained nearly onstant when 1-min stimulations were presented every 20 min. Habituation also depended on the duration of the high K+ stimulations. Five-minute stimulations at a frequeny of every 10 min led to a derease in the response that was twie as fast as did 1-min stimulations applied at this same frequeny. The response was onstant with 5-min stimulations every 40 min. To orret for the possibility that the derease in [3Hlnorepinephrine release arose artifatually from the depletion of ellular radiolabel, the seretion data shown here have been orreted for ongoing loss of ellular radiolabel, determined by lysing the ells at the end of eah experiment and measuring their ontent of [3H]norepinephrine. Also, the intraellular ontent of radiolabel was depleted in onstant proportion with the total intraellular store ofateholamine, whih was assayed by standard biohemial proedures. Finally, ontrol measurements showed that the released radiolabel ohromatographed with genuine norepinephrine. Thus, the diminishing size of the response peaks aurately reflets a derease in norepinephrine seretion. To test the duration of habituation, prolonged rest periods were allowed during repetitive K+ stimulation (Fig. 3). In a typial experiment, high K+ stimulations were presented every 5 min until over half of the ellular response had been lost through habituation. The ells were then allowed to rest for 20 min, after whih a signifiant restoration in the response was found. Subsequent repetitive stimulation led to f f f f T f f I Tf f T f [ f *, N! e 1.0 a 4 Q* 4 e Pro. Natl. Aad. Si. USA 87 (1990) Time after first stimulation (min) FIG. 2. Size of [3H]norepinephrine response peaks depending on the frequeny and duration of depolarizing stimulation. Five experiments are summarized on a sale normalized to the size of the first response peak in an experiment. ah point represents the area beneath a deteted peak of norepinephrine eliited by high K+. *, *, and *, stimulations of 1-min duration; A and o, stimulations of 5-min duration. Frequenies of stimulation in the five experiments were as follows: e, stimulation every 5 min (data replotted from Fig. 1); A, A, stimulation every 10 min; o, stimulation every 20 min; and *, stimulation every 40 min. further habituation. A partial inrease of the response was again observed after another 20-min rest interval was allowed. a2+ and Phorbol ster Disruption of Habituation. The dihydropyridine drug Bay K 8644 (16), an ativator of voltage-gated a2l hannels, was tested for its ability to disrupt habituation. Appliation of Bay K 8644 to habituated ells inreases seretion (Fig. 4). Here, Bay K 8644 was applied ontinuously, beginning at the time of the fifth stimulation by high K+, and the inrease in seretion was rapid in onset. This and the next two responses to high K+ were restored in size to within the range of the initial peak size. It appears, then, that inreased a2+ entry into the ell, due to this hannel ativator, an overome habituation. The seretory response size then dereased with further high K+ stimulation, despite ontinued exposure to Bay K 8644 (Fig. 4), the derease likely being due to a detrimental effet of Bay K 8644 beause exposure to this agent beyond the time shown 3L 2 2 z FIG. 1. [3H]Norepinephrine sereted from P12 ells with repetitive depolarizing stimulation. [3H]Norepinephnrne-loaded ells were plaed in a perfused hamber (see xperimental Proedures) and stimulated with high K+, as shown by arrows and horizontal bars. High K+ stimulations were presented for 1 min every 5 min [3H]Norepinephrine sereted from P12 ells with re- FIG. 3. petitive depolarizing stimulation showing the effet of rest. High K+ stimulation was for 1 min every 5 min, exept at two intervals when the period between stimulations was 20 min.

3 Neurobiology: MFadden and Koshland K+V V V V VF V V Pro. Natl. Aad. Si. USA 87 (1990) 2033 F F Vr F F F F k w S.._ ).= * a= 0) o_ z 0 r_ ).,. _ L- o, P0. I z.- a a. z xa o. FIG. 4. ffet of the a2+ hannel-speifi drug, Bay K 8644, on [3H]norepinephrine seretion during repetitive depolarizing stimulation. [3H]Norepinephrine-loaded ells were stimulated with high K+ for 5 min every 10 min, as shown by arrows and horizontal bars. Bay K 8644 (1,uM) was applied to the ells at the time shown by the bold arrow and was present for the remainder of the experiment, as shown by the shaded horizontal bar. resulted in a preipitous derease in seretion that was not restored by removal of the drug. Phorbol esters were found to inrease the ellular response in a variable manner, depending on whether the ells are habituated or not. When ells were first habituated by repetitive depolarizing stimulation, a transient appliation of PMA resulted, within 5 min, in an inrease in the size of the response of =50% over that expeted had habituation been unaffeted (Fig. 5A). The response size then dereased again with further high K+ stimulation, dereasing to the previously habituated size within '60 min of the PMA appliation. In the same experiment, a seond dose ofpma was applied 90 min after the first, and the response was only slightly inreased (Fig. 5A). The failure of a seond dose of PMA to inrease the response as effetively as the first shows that simple washout of the first dose is not the reason for the eventual derease in the response. PMA interferes with later development of habituation (Fig. 5B). When PMA was applied to ells before frequent depolarizing stimulations, no habituation was observed for over 30 min, and the response size then diminished only slightly over the next 60 min. Prior treatment with PMA an thus inhibit the habituation, ausing ells to serete nearly onstant amounts of norepinephrine, with a pattern of repetitive high K+ stimulation that would otherwise produe very obvious habituation. To test the possibility of a reiproal effet, an experiment was performed in whih PMA was first applied to ells and high K+ stimulations were then presented suffiiently infrequently that little habituation would be expeted. An unusual pattern of seretion was observed (Fig. 5), in whih the size of the seretory response inreased from the first to the seond high K+ stimulation by =80% and then remained at this high level for the next hour. This result shows that the effets of PMA ontinue to build for at least 20 min in the absene of habituation. PMA is thus most effetive in inreasing seretion in the absene of habituation. DISUSSION Autonomous ellular Habituation. These studies indiate that neuronally differentiated P12 ells are apable of habituation, often termed the simplest form of learning. Habituation has previously been studied in ellular detail in many model systems, inluding vertebrates (17, 18); invertebrates (19-21); polysynapti pathways suh as the mam- a 0 O- _ T- o ab a. LD 0 _-. z 0 0 ) K+ f I f f I f f I f f f f f f f f f f f f 60 FIG. 5. ffet of PMA on [3H]norepinephrine seretion with high K+ stimulation at the times shown by arrows and for the durations shown by horizontal bars. PMA (0.1 AtM) was applied to the ells for 2 min at the times shown by bold arrows. (A) PMA applied during frequent stimulation; high K+ stimulation was for5 min every 10 min. (B) PMA applied before frequent stimulation; high K+ stimulation was for 1 min every 5 min. () PMA applied before infrequent stimulation; high K+ stimulation was for 1 min every 20 min. malian spinal reflex (22); monosynapti pathways in molluss (3, 23, 24); rustaeans (25); and monosynapti reflex pathways in amphibians (26). In Aplysia, homosynapti depression through dereased a2' entry into sensory neurons has been proposed as a mehanism of habituation (27). In eah of the above systems, the presene of ell-ell interations has left open the question as to whether suh a memory an our in the single ell, in ontrast to ommuniation between ells. Synapti input between ells, as the mehanism of habituation in P12 ells, seems unlikely, beause few ell-to-ell synapses are seen in mirosopi examination (5), and it would therefore have to be argued that any suh synapses would have enormous quantitative effets. Any diffusible regulatory moleule sereted by the ells into their ulture

4 2034 Neurobiology: MFadden and Koshland medium would have only autoregulatory effets beause of the uniformity of the ells. The habituation of P12 ells desribed here thus reflets autonomous biohemial regulation of the single ell. While several terms may be used to desribe a derease in a response (desensitization, adaptation, depression, inhibition), the term habituation seems most appropriate for the observed ellular behavior beause it expliitly serves to desribe hanges in behavior with repeated stimulation. Nine riteria for habituation in omplex organisms have been enumerated by Thompson and Spener (12, 19), and we have evaluated the behavior of P12 ells with respet to several of these. The primary riterion of dereased responsiveness with repetitive stimulation is learly observed when ells are repetitively stimulated by high K+ medium. A seond riterion, inreased habituation with inreased frequeny of stimulation, is also met by repetitively stimulated ells. This frequeny dependene shows that the dereasing response is not simply due to deterioration of the ells in the ourse of the experiment. A third riterion, reovery of responsiveness when stimulation is withheld, is also a property of these ells. At least part of the ellular responsiveness is reovered with a rest period, showing that an endogenous mehanism operates between stimulations in opposition to the down-regulation of seretion with habituation. Responsiveness that is not restored with a rest period does reflet a long-term downregulation of seretion. Alternatively, P12 ells may demonstrate a fourth riterion of habituation that is observed in animal systems-i.e., prolonged reovery times aused by depressing a system below its habituated level (12). A fifth riterion for habituation that was met by the ells was dishabituation of the response by a heterologous signal. A ommon manipulation in animal studies is the presentation of a seond form of stimulation that interrupts habituation, often by inreasing the sensitivity of the system (28). PMA and Bay K 8644, ativators of protein kinase and of voltage-gated a2+ hannels, respetively, were shown to dishabituate ells. The period of information storage, an important feature of any memory mehanism, was dedued on the basis of the longest interval between repetitive stimulations that ould produe habituation. The derease in ellular responsiveness in single P12 ells an persist for up to -20 min. That length of time is harateristi of short-term memory for some organisms and long-term memory for others. The similarity of habituation in P12 ells and in the model systems studied by others does not indiate that all biohemial systems will be similar but does indiate that priniples of information proessing may be similar in these different types of systems. The method we have used to depolarize ells, delivery of external high K+, does not allow as rapid and preise variation of membrane voltage as do eletrophysiologial voltage lamp methods. On the other hand, the method has the lear advantage of being free of feedbak effets from postsynapti ells, simplifying the interpretation of the effets of externally applied agents. Interplay of Seond Messenger Systems and ellular Habituation. ells are dishabituated by inreased a2l entry and by PMA treatment, indiating that known seond messenger systems an oppose the ellular mehanism of habituation. Bay K 8644 is a dihydropyridine derivative known to ativate L-type a2+ hannels in numerous preparations (16, 29, 30). Other laboratories have deteted this hannel in P12 ells, though it is reportedly present in redued numbers after nerve growth fator treatment for several weeks (31-33). In our work, whih involved ulturing ells for 1 week with nerve growth fator, this hannel appeared to ontribute to most of the depolarization-mediated seretion by these ells, Pro. Natl. Aad. Si. USA 87 (1990) as shown by the omplete bloking of seretion by verapamil (data not shown), an antagonist of the same lass of hannels. a2l influx is a requirement for depolarization-mediated neuroseretion in P12 ells. A simple interpretation of dishabituation of P12 ells by Bay K 8644, then, is that inreased a2' entry into the ell due to this agent an ompensate for a down-regulation of a21 signaling during habituation. Down-regulation of a2+ urrent in nerve endings has been proposed as a mehanism of habituation in Aplysia (27, 34). A major question now is how the a2+ signal may be down-regulated and how this information is stored between depolarizing stimulations. PMA ativates seretion in a variety of a2+-dependent systems, presumably through its ativation of ellular protein kinase. It has been proposed that the ation of PMA involves regulation of the intraellular a2+-sensing mehanism involved in seretion beause PMA raises the a2+-sensitivity for seretion in ells made permeable to a2+ (10, 35-37). However, preise interpretation of how PMA affets the habituated ell is ompliated by the fat that many proteins are substrates for protein kinase and many proesses may, therefore, be affeted by ativation of this kinase (38, 39), inluding a blokade of inward a2+ urrents (40) that ould be important in modulating seretion from habituated ells. A further ompliation is the possibility that PMA may not be absolutely speifi as a protein kinase -ativating agent: dereased a2+ urrents due to a protein kinase -independent ation of phorbol esters have been reported, though these measurements were made at a 100-fold higher PMA onentration than used here to dishabituate P12 ells (41). 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