Proc. Natl Acad. Sci. USAVol. 76, No. 11, pp. 5987-5991, November 1979Neurobiology

Chronic treatment with lithium or desipramine alters dischargefrequency and norepinephrine responsiveness of cerebellarPurkinje cells

(antidepressants/catecholamines/locus ceruleus/iontophoresis/ft receptors)

G. R. SIGGINS* AND J. E. SCHULTZt*Arthur V. Davis Center for Behavioral Neurobiology, The Salk Institute, San Diego, California 92138; and tPharmazeutisches Institut, University of Tubingen,Morgenstelle 8, 7400 Tubingen 1, Federal Republic of Germany

Communicated by Floyd E. Bloom, August 8, 1979

ABSTRACT Cerebellar Purkinje cells were studied byelectrophysiological techniques in rats treated chronically witheither desipramine (DMI) or lithium chloride given intragas-trically. A striking decrement occurred in discharge frequenciesof simple spikes and climbing fiber bursts in both groups ofanimals, similar to the depression produced by iontophoresisof these agents. Chronic treatment with DMI markedly de-creased responsiveness to iontophoretically applied norepi-nephrine (NE), whereas long-term LiCl therapy slightly en-hanced response to NE; responses to 'y-aminobutyric acid wereunchanged by these treatments. The inhibitory responses tolocus ceruleus stimulation were unaffected by chronic LiCltreatment. The effects of these chronic treatments on respon-siveness to NE are opposite to the effects these same drugsproduce when administered by acute iontophoresis to singlecells: DMI then potentiates and LiCl antagonizes noradrenergicresponses. These results provide electrophysiological evidencefor reciprocal adaptive changes in NE sensitivity, supportingresults of biochemical studies.

Lithium chloride, lithium carbonate, and tricyclic dibenza-zepines such as imipramine and desipramine (desmethylimi-pramine, DMI) have proven to be effective antidepressantagents in clinical studies (1-4). Although the exact mechanismof their therapeutic action remains obscure, acute treatmentwith LiCi is known to exert biophysical (e.g., ref. 5) and neu-rochemical (3, 4, 6, 7) actions, including antagonism of nor-epinephrine (NE)-induced increases in cyclic AMP (8,9). Singleacute doses of imipramine or DMI rapidly block uptake ofcatecholamines into adrenergic terminals (10-12), a findingwhich contributed to the catecholamine theory of depression(13).

These antidepressant agents have not yet been well studiedelectrophysiologically. Iontophoretic administration of Li hasbeen reported to antagonize inhibitory adrenergic responsesin both hippocampal pyramidal neurons (14) and cerebellarPurkinje cells (15), a finding consistent with the mediation ofthese catecholamine responses by cyclic AMP (8, 9). Results ofphoretic tests of the tricyclic antidepressants have been moreequivocal. DMI potentiates NE responses in the cerebellarPurkinje cell (16) and cortical neurons (17), thus supporting theuptake-blocking action of DMI. Other cortical studies showeither a dual effect (e.g., ref. 18) or a pure antagonism of NEresponses (e.g., ref. 19).

Clinical therapy with either Li or the tricyclic antidepressantsusually requires several days of drug treatment (2-4, 13), incontrast to the rapid onset of amine uptake or receptor block-ade. This discrepancy might be clarified by recent observationsof the adaptive (reciprocal) changes demonstrated for NE-

activated adenylate cyclase and adrenergic receptors in animalschronically treated with drugs known to alter functional cate-cholamine levels. Thus, chronic but not acute treatment withimipramine or DMI decreases the sensitivity of mesolimbic (20)and cortical (21) adenylate cyclase to stimulation by NE whiledecreasing f-receptor binding (22, 23). In iontophoretic studies,chronic DMI treatment altered responsiveness to 5-hydroxy-tryptamine (24), and chronic Li treatment appeared to blockthe DA supersensitivity elicited by chronic haloperidol (25).The aim of the present study was to compare the effects of

chronic Li and DMI treatment on the physiology of the ratcerebellar Purkinje cellM, which exhibits inhibitory noradren-ergic responses that are likely to be mediated by cyclic AMP(see ref. 26) and that are affected in opposite directions byphoretic application of Li or DMI (15, 16).

METHODSA total of 25 adult albino rats were used, with five groups in theDMI study. The first group ("chronic DMI, high") received,by gavage, DMI-HCl at 50 mg/kg in 2 ml of tap water oncedaily for 3 days, and then 30 mg/kg per day thereafter for 7-11days. The second group ("chronic DMI, low") received DMIat 30 mg/kg per day for 8-18 days. These two "chronic groups"were tested electrophysiologically 24-36 hr after the last dose,when cerebellar DMI is roughly one-half the level duringtreatment (unpublished data). The third group ("controls")received 2 ml of tap water by gavage daily for 6-10 days. Thefourth group ("DMI withdrawal") received DMI daily for 10days and then treatment was terminated for 5 days beforetesting. In the fifth group ("acute DMI") animals received onlyone dose of DMI (30 mg/kg) by gavage prior to anesthesia andtesting. The intragastric route of administration was chosen tomaintain fairly constant brain levels of DMI and to avoid theperitonitis and resultant disruption of drug absorption oftenassociated with chronic intraperitoneal injection.

Four groups of animals were studied for Li effects. In the firstgroup ("chronic Li"), LiCl at 60-75 mg/kg was administeredonce per day in 2 ml of water, by gavage for 9-12 days sup-plemented by ad lib intake of 10-15 mg/kg per day in thedrinking water which also contained 0.9% NaCl. This groupwas tested electrophysiologically 2-12 hr after the last dose ofLiCl, when serum Li levels were equal to those during treat-ment (unpublished data). A control group of rats received NaClat 75 mg/kg per day by gavage as well as 0.9% NaCl in thedrinking water. A third group ("Li withdrawal") received LiCI

Abbreviations: DMI, desipramine; NE, norepinephrine; ISH, interspikeinterval histogram; GABA, 'y-aminobutyric acid; CF, climbing fiber;DA, dopamine.f G. R. Siggins and J. E. Schultz (1978) Abstracts 7th InternationalCongress of Pharmacology (IUPHAR), p. 346.

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5988 Neurobiology: Siggins and Schultz

daily but at day 10 treatment was terminated for 5-7 days be-fore testing. A fourth group ("acute Li") was given a singlegavage of LiCI at 75 mg/kg 2-12 hr before anesthesia andtesting. The chronic group had plasma Li levels in the 0.5-1.3meq/liter range (measured 0-24 hr after the last dose), as

measured by flame photometry; growth weight was equal tothat of the controls and only rare cases of overt diarrhea or Litoxicity occurred.The animals weighed 165-310 g at the time of electrophys-

iological testing. Anesthesia was induced with either chloralhydrate (25300 mg/kg) or 3% halothane and then maintainedthroughout the experiment with halothane (0.75-1% in air)alone. The methods of stereotaxis, craniotomy, and surgicalpreparation for neuronal recording have been reported (15,16).For recording discharge patterns and rate, we used single-

barrel pipettes (tip size z 1 um, 2-5 MQ; 3 M NaCI) insertedinto areas 6 and 7 of the vermis. Recording from and identifi-cation of single Purkinje cells were performed by standardelectrophysiological methods as described (15, 16). Spike trainswere stored on magnetic tape (bandpass, 30 Hz to 21 kHz);traces of spike frequency (digitized pulses integrated over 1-secintervals) as well as unfiltered d.c. records from the pipette tipwere recorded on polygraph paper.

Firing frequency was quantitized on-line with a PDP-11computer, using interspike interval software developed by K.Liebold (FISH.MAC; DECUS, 11-320). To generate interspikeinterval histograms (ISHs), spikes (magnitude, 0.4-2.0 mV;bandpass, 600-6000 Hz) were gated and converted to square

pulses for input to the PDP-11. The ISH program identifies andquantifies the complex climbing fiber (CF) burst by the oc-

currence of two or more spikes within 3-7 msec. Several pre-

cautions (27) were taken to verify that the bursts recorded arose

from CF activation and did not represent noise, injury dis-charges, or spikes from neighboring cells.

Iontophoresis and appropriate controls were accomplishedby means of five-barrel pipettes (tip diameter, 5-8 ,um) (15, 16,28, 29). Pipette barrels were filled with: DL-norepinephrine-HCI0.5 M, pH 4; y-aminobutyric acid (GABA) 0.5 M, pH 4; NaL-glutamate, 0.5 M, pH 8. To facilitate quantification of agonistsensitivity, an effort was made to decrease intrapipette vari-ability in drug release by using an alternating high/low currentthreshold technique designed to optimize drug ejection froma given pipette. The pipette was "warmed up" by passing manyregularly repeating pulses (20-60 sec in duration) of a givenagonist, alternating between a fixed high and a variable lowcurrent; each time, the lower current was decreased. The highcurrent acts to warm up the barrel for the subsequent low "test"pulse. The low current was decreased until a response was no

longer seen and then was returned to that current which justproduced a threshold response (15-20% of baseline). To mini-mize pipette variability further, tests of discharge patterns andresponsiveness to monoamines by phoresis were carried out inabout half the experiments by a single-blind, tandom-animal

protocol, whereby a coded control animal and a coded,chronically treated (Li or DMI) animal were both prepared on

the same morning in separate stereotaxic frames within thesame recording set-up. Single-blind recording of spontaneousactivity and iontophoresis was performed in one animal withone penetration of the cerebellum (to 5 mm deep), followed byelectrode withdrawal and immediate penetration of the cere-bellum of the second animal; thus many cells (up to 5-10 cellsper pass, 15-40 per animal) were recorded in an alternatingfashion with the same single- or multibarrel pipette.

RESULTSNeuronal Discharge Patterns. A striking finding in this

study was the low discharge activity of Purkinje cells in ratschronically treated with DMI or Li. Our initial objective was

to use a "blind" protocol to compare neuronal firing and re-

sponses to iontophoretically applied drugs in chronically treatedand control rats. However, the extreme slowness or frank ab-sence of spontaneous firing in the high-dose DMI rats imme-diately revealed the rat's treatment history. The 56 neurons

observed in six rats chronically treated with DMI at the highdose showed mean Purkinje firing rates less than one-fourththose of the six control rats studied (Table 1). The chroniclow-dose DMI group also showed significantly decreased rates.Even a single dose (30 mg/kg) of DMI (four rats) significantlydecreased spontaneous firing, although less so than with chronictreatment. Purkinje firing returned to control rates within 5days after withdrawal of treatment (four rats).

These depressive effects were further characterized bycomparing ISHs from Purkinje cells of control, chronic DMIhigh, and DMI withdrawal groups (Fig. 1). A much greaterpercentage of interspike intervals exceeded 75 msec in thechronic DMI, high rat compared to the control, although themodal peaks (the interval with the largest number of simplespikes) for the two populations are similar. A similar promi-nence of long interspike intervals occurs during the NE-evokeddepressions of firing in normal animals (16). However, in thepresent study, many Purkinje cells (identified by their CFcomplex spikes) displayed few single or simple spikes in thechronic DMI, high group, in marked contrast to the high ratesin control and DMI withdrawal rats. The ISH program alsodetected a significant reduction in the frequency of CF burstswith acute and chronic DMI (high dose and low dose) treatmentbut not in DMI withdrawal animals (Table 1).

Chronic administration of Li also significantly decreased theaverage single-spike and CF-evoked activity of Purkinje cells(Table 2). Purkinje cells of animals withdrawn from chronicLi treatment fired at control frequencies for simple and CFactivity. Although cells of acute Li animals displayed normalsimple-spike firing rates, the mean frequency of CF bursts inthe same animals was significantly decreased.

Although the mean rates were significantly decreased in bothchronic treatment groups, several individual Purkinje cells inthe chronic Li group and in the chronic DMI, low group dis-

Table 1. Effects of intragastric DMI on discharge frequency of cerebellar Purkinje cells

Chronic DMI DMIProperty Controls* Acute DMI High dose Low dose withdrawal

Simple-spike firing rate,spikes/sec . 30.2 (53) 21.St (41) 7.54 (56) 16.1$ (39) 83.3 (57)

CF bursts persec 1.22 (49) 0.52t (42) 0.38$(56) 0.388(38) 1.09 (55)

Data are mean values. Number of cells tested is shown in parentheses.* DMI and Li controls combined.t P < 0.05, 1-way analysis of variance, Newman-Keuls analysis (compared to controls).P < 0.01.

Proc. Natl. Acad. Sci. USA 76 (1979)

Neurobiology: Siggins and Schultz

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Fic. 1. Effects of chronic intragastric DMI on firing rates and patterns of cerebellar Purkinje neurons. Representative ISH from a controllPurkinje cell (A) is compared to that of a Purkinje cell (upper and lower right) from a high-dose DMI-treated animal (B and D) and to that froman animal from the DMI withdrawal group (C). Shortest intervals (high interburst rates) for peaks of climbing fiber bursts (asterisks) and narrow

modal peaks of' simple spikes (peak <20 msec) are typical of control and withdrawal animals, compared to the paucity of climbing fiber burstsand the broad simple-spike peak in chronic DMI animals. B and D display the same ISH, but the abscissa in B is expanded to accommodatethe abnormally long intervals (slow firing pattern) of the DMI-treated cell. (Insets) Ratemeter records (spikes converted to square pulses andintegrated over 1-sec intervals) taken from each of the three cells.

played spontaneous simple and CF activities indistinguishablein rate and pattern from those of control animals.

lontophoresis. Responses of Purkinje cells to phoretic NEwere compared in the various groups. However, because manyof the cells of the chronic DMI, high group were firing at ab-normally low spontaneous rates, it could be argued that thesedecreased rates could directly alter NE responses. Therefore,in about one-third of the cells the firing rate was increased tonormal levels by the continuous passage of low currents of L-glutamate. In general, spontaneous or glutamate-evoked firingin DMI- or Li-treated rats was readily depressed by NE, al-though occasionally some neurons in chronic DMI-treated ratsshowed a breakthrough of firing after an initial short depression

Table 2. Effects of Li on discharge of cerebellarPurkinje cells

Acute Chronic LiProperty Controls* Li Li withdrawal

Simple-spike firing rate,)spikes/sec 30.2 30.5 13.3t 29.6

(53) (39) (82) (37)('F b)ursts 1.22 0.63t 0.47t 1.13

per sec (49) (32) (76) (32)

Data are mean values. Number of cells tested is shown in paren-leses.l)MI and Li controls combined.

t)P< 0.01, 1-way analysis of variance, Newman-Keuls (compared to( ontrols).

in spite of continued application of NE. More importantly, themean threshold NE current required to depress neurons ofchronic DMI high rats was significantly higher than that forcontrol cells (Table 3). In cerebella of acute DMI and DMIwithdrawal animals, mean NE thresholds were not significantlydifferent from control values.

Chronic treatment with LiCI decreased NE thresholdscompared to controls (Table 3; P = 0.02). Although acute Litreatment did not significantly alter mean NE thresholds, thosein the Li withdrawal group were still depressed 5 days aftercessation of chronic Li treatment. However, because of thesmall number of cells studied in this withdrawal group, thisdifference must be interpreted with caution.

Table 3. Effects of intragastric DMI or Li on iontophoreticcurrents of NE required for threshold inhibition of cerebellar

Purkinje cellsAcute Chronic

Drug Controls* treatment treatment Withdrawal

DMI 24 + 2.7 16 + 2.9 43 + 3.6t 31 + 5.9(26) (18) (28) (22)

LiCl 24 + 2.7 17 i 3.3 14 i 2.61 14 + 2.41(26) (8) (43) (13)

Data are shown as mean (+SEM) nA; number of cells is shown inparentheses.* DMI and Li controls combined.t P < 0.01, 1-way analysis of variance, Newman-Keuls, or Student's

t test.P = 0.02, Student's t test.

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5990 Neurobiology: Siggins and Schultz

As a control for the specificity of these changes in NEthresholds induced by the drugs, Purkinje cells were also testedfor thresholds to GABA. No significant differences were seenbetween the mean GABA threshold currents of the various DMIand Li treatment groups studied (all P > 0.1; n, 6-30).

Stimulation of Locus Ceruleus. Because phoretic Limarkedly antagonizes inhibitory responses of Purkinje cells tostimulation of the locus ceruleus (15), an attempt was made todetermine if this blockade persisted after chronic Li treatment.Stimulation of the locus ceruleus and relevant analysis wereaccomplished as described (15, 29). Twenty Purkinje cells weretested in chronic Li animals in which subsequent histologicstudy verified precise placement of the stimulating electrodein the locus ceruleus nucleus. Of these, 16 cells (80%) respondedwith marked inhibitions of spontaneous firing to trains of stimuliat 10 Hz; the rest were unresponsive. Threshold voltages in thesecells (range, 2-30 V; mean, 12.7 V) were near those recordedfor normal animals under identical conditions (15, 29). Another17 Purkinje cells were studied in animals in which the stimu-lating electrode passed outside but within about 0.5 mm of thelocus ceruleus: 10 (59%) of these cells displayed clear inhibitions,at higher thresholds (range, 7-35 V; mean, 22.2 V); the re-mainder did not respond.

DISCUSSIONAn unexpected finding of this study was the marked decreasein the rate of Purkinje cell firing in animals chronically treatedwith DMI or Li. Such action resembles the depressions in firingproduced by iontophoresis of both these agents (15, 16) and maysuggest that Purkinje cells do not become tolerant to these directdepressant actions. However, the question persists as to whetherthe chronic depressions in firing represent direct action of theantidepressant drugs by extracellular or intracellular accu-mulation or some other (perhaps compensatory) mechanismarising indirectly from the treatment. Biochemical studies (ref.30; unpublished data) show persistent, measurable accumula-tion of DMI in brain during equivalent chronic DMI or im-ipramine treatment, as well as continued antagonism of cate-cholamine uptake. In our Li-treatment protocol, serum levelsof Li also steadily accumulate to levels approaching therapeuticserum levels in depressed patients. An additional study withchronically treated (Li or DMI) animals on withdrawal for only2-3 days rather than 5 days (see ref. 31 for a description of thetime course of the biochemical effects of DMI treatment andwithdrawal) might reveal whether the depressions in firing rateare present when circulating or brain drug levels are negligible,thus testing for direct effects of the drugs.

It is feasible that the severe depression of Purkinje cell firingseen in animals treated with the high-dose schedule of DMI (50mg/kg per day for 3 days; final dose, 30 mg/kg per day) arisesas a result of excessive medication and possible sedative side-effects. However, the fact that these rats behaved normally andgained weight at the same rate as controls suggests normal dailyactivity. Although the low-dose DMI schedule (30 mg/kg perday) gave somewhat higher mean firing rates, significant de-pression of firing still occurred. The response curve of dailyimipramine dose versus NE-generated cyclic AMP in cerebralcortical slices indicates that just-maximal biochemical effectsare seen at daily intragastric doses of 20-30 mg/kg per day(unpublished data). Because our initial aim was to use an opti-mal dose for clear-cut detection of physiological effects, wechose the maximal dose, 30 mg/kg per day. Given the lesserabsorption by the intragastric route, this single daily dose iscomparable to the multiple daily intraperitoneal doses totaling10-20 mg/kg per day used by others (cf. refs. 20, 22, 30, and31).

It is possible that depression of firing represents an interactionwith the anesthetic. However, the depressions elicited bychronic DMI in anesthetized animals can be prevented bypretreatment with the neurotoxic agent 6-hydroxy dopamine(unpublished data). Therefore, anesthetic or toxic factors cannotbe solely responsible for the depressant action; an effect in-volving accumulation of catecholamine may be implicated.

Iontophoresis was used to assess the sensitivity of Purkinjecells to NE. We detected a significant increase in the meanthreshold iontophoretic current required for depression ofPurkinje cells in animals chronically treated with DMI and adecrease in thresholds in chronic Li animals. The results withDMI contrast with those of Montigny and Aghajanian (24), whofound no significant change in the NE responses of rat hippo-campal neurons with chronic tricyclic antidepressants. Thisapparent discrepancy may arise from differences in brain re-gions studied or in the methods used to assess NE sensitivity: weattempted to decrease intra-pipette drug-release variability bymeasuring response thresholds with alternating high and lowdrug ejection currents, the same pipette, and a tandem ani-mal/single-blind protocol, whereas Montigny and Aghajanianused a protocol based on the time and current required to obtaina 50% decrease in firing rate.Our finding of decreased NE responsiveness in chronic

DMI-treated animals is likely to be an electrophysiologicalmanifestation of the subsensitivity of the brain NE/cyclic AMPsystems (20, 21) and decreased f-receptor binding (22, 23) seenin vitro. As such, it may represent an adaptive homeostaticmechanism in response to the increasing functional levels ofextracellular NE produced by blockade of reuptake. Thesehigher NE levels may be sufficient to overcome the subsensi-tivity to NE, to the degree that depression of discharge results.This NE subsensitivity contrasts with the moderately enhancedresponsiveness to phoretic NE elicited by chronic Li treatment.The latter effect may also represent an opposing adaptivemechanism because acute Li treatments antagonize neuronaldepressions evoked by NE and locus ceruleus stimulation (14,15), as well as NE-activated adenylate cyclase (8, 9). The lackof effect of acute intragastric administration of Li on NEthresholds, in contrast to the NE blocking action of phoretic Li(15), may result from initial low serum Li levels produced bythe intragastric route, compared to high extraneuronal Li levelsgenerated by iontophoresis.The present findings taken with previous biochemical studies

suggest that acute responses of neuron systems may not be anadequate basis for hypotheses of the cause of the affectivepsychoses that respond to chronic antidepressant treatment.Moreover, the effects of antidepressant drugs on other neuro-transmitter systems such as those of 5-hydroxytryptamine (6,24) and dopamine (7, 25) may support a multifaceted etiologyof depression. The present results also support the possibilitythat direct alteration of cell firing per se could serve an anti-depressant function. Such a view is compatible with the hy-pothesis that manic-depressive disorders represent alterationsin cellular ionic permeability (4, 32, §), which chronic Litreatment serves to restore toward normal. Finally, it is alsofeasible that adaptive receptor changes could combine withdirect effects of the antidepressant drugs; these concerted ac-tions might then provide the mechanisms of therapy on the onehand or undesirable side-effects on the other.We thank J. Schulman, D. Games, and F. McCoy for technical as-

sistance, K. Liebold for his computer expertise, and N. Callahan for

§ J. Mendels, T. A. Ramsey, and A. Frazer (1977) Scientific Proceed-ings of the 130th Annual Meeting of the American PsychiatricAssn., p. 150.

Proc. Natl. Acad. Sci. USA 76 (1979)

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secretarial skills. We are indebted to Drs. F. Bloom, B. Hoffer, and S.Henriksen for helpful criticism of the study and the manuscript.Desipramine was a gift of the USV Pharmaceutical Co. This study wassupported by grants from the National Institute of Mental Health(MH-29466), the Volkswagen Stiftung, and the Alexander-von-Humboldt Stiftung.

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Neurobiology: Siggins and Schultz