COGNITIVE BRAIN RESEARCH CognitiveBrain Research1 (200i) 000-000 www.elsevier.com/ locate/bres

Short communication 3 Mental stimulation increasescirculating CD4-positive T lymphocytes: a preliminary study

5 MarianC. Diamondu'*,Jeanne Weidneru, Peter Schowb't, Stanley Grellb, Marian Everett"

6 "DepartmentorIntegratiue Bioti#"iif',Iiii,ll"::{;,';:,::::,:":f';:,:,ry:;: cAe1720'3140' usA 7 ;?,;!,:;'!"i"ii;#,'!,:!iv' 8 "Department of the Alumni Association, University of Califurnia, Berkeley, CA 94720, USA

9 Accepted 17 April 2001

0 J' I Abstract "' =,,,u,,,.ji',. 2 To stimulate the dorsolateral frontal cortex, 12 healthy, adult, human females played contract bridge for t.S tr between initial and final l blood sample collections. FIow cytometric analyses of samples, performed in triplicate, showed a signijbant incdase in CD4-positive T 4 lymphocytes. The dorsolateral frontal cortical thickness is significantly and bilaterally reduced in immun6iin-ornpbtent female, nude mice. 5 Thymic transplantsreverse the deficient cortical thicknessand CD4-positive cell numbers. O 2001 Published by Elsevier ScienceBV.

Theme:Neval basisof behavror .i' ,,,'r-!,;1,, tt' Topic: Cognition .,,',t ' , ,, ',,,, 1.,,.,,, '8 Keywords: Immune incompetent female nude mice; Dorsal lateral frontal codex; CD4j)ositive Tlymphocytes; Stimulation :9

0 Many investigators have clearly demonstrated that ma- Es3endelly this project demonstrated that the dorsolateral 4 t nipulation of the highest cognitive region of the brain, thc frorrtal cortex was bilaterally deficient, as measured by 4 2 neocortex, has a role in immune regulation. Particular microscopic thickness [10], in the female, immune in- 5 3 alteration of one system can subsequently affect tne:O'tfi"br. competent, nude mouse when compared with the cortical 5 + In the early 1980s lesion studies of the cerebral:icortex thicknessof an immune competentmouse from the BALB/ s 5 resultedin enhancingor inhibiting effects on the immune c strain. In 1996 and 1991, two more studies Ul,Izl 5 6 system [1,14]. Ablation of the left dorsal and lateral confirmed this cortical deficiency and in addition demon- 5 z frontal, parietal and occipital cortical .ardas i ... dents strated that cortical and blood immune deficiencies in the s 8 causeddecreased activities in T cells and natural killer nude mouse could be reversedwith thymic transplants. 5 g cells. Lesions in the contralateraLcoftlcal eas increased All of these studies were performed on female nude or 5 0 the Sameimmune cells. it,,,. BALB/c mice. Results obtained from female C3H mice 5 t Inspired by these initial inVestigAtions,we studied the have been partially replicated in male mice for NK cell 5 z congenitally athymic nude rnouse {o identify areas of the activity [6] and for lymphocyte proliferation in female 6 3 cerebral cortex that may'be affected by the T cell-deficient Sprague-Dawley and male Wistar rats [2,13]. These 6 4 state.Nude mice h3ve.few or no CD4-positive T lympho- findings with rodents suggest that cortical immune re- 6 5 cltes. sponses can be generalized across both sexes and in 6 6 In 1986, we" publi$ed [9] our first research project different speciesand possibly in human beings. 6 z dealing with'the berebral cortex and the immune system. The functions of the dorsolateral frontal cortex in 6 humans include working memory, changing set,judgment, 6 initiative, planning ahead, sequencing data, etc. Some 6 xCorresponding +l-510-642-4547; +1-510-643- author. Tel.: tax: investigators have utilized the xenon dynamic single 6 6264. photon (SPECT) E-mailaddress : [email protected] (M.C. Diamond). emission-computedtomography during 6 'Presentaddress: Genentech, Inc,, I DNA Way #50, SouthSan Fran- the performance of the Wisconsin Card Sorting Test j cisco,CA 94080,USA. (WCST) to demonstrate activation of the dorsolateral j

0926-61101011$- see front matter @ 2001 Publishedby Elsevier Science BV PII: S0926-6'110(01 )00059-3 M.C. Diamond et al. I Cognitive Brain Research I (2001) 000-000

fiontal cortex in humans [6]. The WCST is a problem- Table I 7 Mean values for percentage of antigen-beanng lymphocytes in paired 1 3 solving, abstractreasoning test used as a sensitiveindicator samples 4 of frontal lobe integrity in humans. Individuals with 1 n Mean pre- Mean post- Mean S,D. 5 dorsolateralprefrontal lesions do poorly on the WCST. Antigen 7 sample sample difference 6 Berman and Weinberger l4l rneasuredregional cerebral 1 '7 7 blood flow using xenon inhalation methods while normal CD3 68.43 6885 0.43 3.30 cD8 t2 27.03 26.98 - 0.04 3.3'7 I 8 subjects performed 10 different tasks including the WCST. CD 56 t2 14.12 14.06 -0.06 3.81 8 dorsolateral prefrontal cortex was activated by these 9 The cD4 t2 49.06 50.90 1.84* 3.53 8 00 tasks.Thus, the findingsof severallaboratories t3-5,7.161 Day I 4 41.41 47.76 0.29 5.93 8 0l have shown that regional activation of the frontal lobes can Day 2 4 s2.07 53.92 1.85xx 0.87 8 02 occur in responseto cognitive challengesproduced through Day 3 41.65 51.03 3.38x* 1.84 E 03 performance of a standard neuropsychological card test. *Wilcoxon signedranked test and sign test P<0.05; pairedltest P<0.10; I 04 In search of a well-known card playing experience. **paired t-test P<0.05. 8 05 insteadof the professionally known WCST, we chose the 06 card game of contract bridge This game has the potential a Wilcoxon signed ranks test and a sign test were used to l 07 to stimulate many of the functions attributed to the determine increases of the cells in the post-sample com- 1 08 dorsolateralfrontal cortex, though it is not an established pared to the pre-sample.A significance level of P<0.05 I 09 scientific way to activate the prefrontal cortex. We hypoth- was used for the analyses. I l0 esizedthat while individuals played bridge, this areaof'the ,rJi. Table I offers the mean values fog'pereentageof antigen I 11 cortex might be stimulated and possibly influence the bearing lymphocytes in paired Fig. 1 presentsthe 1 12 production of T lymphocytes. Therefore, we planned to !.?,q..rp-Jes: mean difference between pailed s-rnples of lymphocyte I 13 take blood samples before and after adult women played antigens. By comparing v9,,.1ousblood samples before and l l4 1.5 h of contract bridge and to quantify the number of T after 1.5 h of bridge p, i g.. aislpnificant increase was I l5 lymphocytes in the blood samples,including CD3, CD4, found in CD4-positiveT lymph"ocytesas measuredby both I tD CD8 and CD56 cell types. the Wilcoxon signeiiiant test (Z:-2.040: P<0.041) and I t7 The following procedures were designed to test our the sign test (P<0.039)"(se" Fig. l). Although there was I i8 initial hypothesis.Twelve, adult, human, female subjects only a trend'ifid-icati.Llgan increase in CD4 antigens as 19 (age range 70-80 years) were easily recruited from the I measuredo^y,,,,the Paired (/(11):1.809; P<0.098), z0 Orinda, California bridge clubs. (Our university Bridge /-test 1 therewas a significantinteraction between day of the study 21 Club had no female students,only males.) Females were I and CD4 increase,with statisticallysignificant increases 22 preferred for two reasons: (1) becausefemale mice were . ..:: I occur,ring.fbr subjects who participated on the last 2 days I LJ usedin the initial studiesby the French scientistsstudying of,the s,triily(Day 2: t(3):4.212' P<0.024; Day 3: r(3): I L4 cortical/immune interactions [4,15]; and (2) becauseall 3.686:P<0.035). Only one subjectwas aberrantfor day l, I zi of our animal studies correlating the cerebral cortex wlthi pieVentinga significant finding for this group. No signifi- 26 the immune system have been carried out on femal"es 1 cant increasesin CD3, CDS or CD56 were detectedas 1 z7 [9,11,12]. The results of our prescreeningquestioiiiiaire measuredby the Wilcoxon signed rank, sign and paired 28 indicated that all the human, female subjects were'free of.! 1 /-tests (see Table 1). In addition, no significant CD4 cell 29 neurological or systemic illnesses with implidatiorls,,,foi l 30 cerebralmalfunction. 31 The ladies were divided into thrce groups of four 2.4 women in each group for card playing. Ten riiilliliters of 33 heparinizedblood were drawn through antisepticskin from 1.5 )+ the median cubital vein in each wornan prior to playing 35 contractbridge for 1.5 h; a this time anothersample of 36 10 ml was collected. Within 2:h of collection, flow 1.0 31 cytometer analysis of sarnples-was performed in triplicate 38 on an EPICS XL_MCL (Beckman-Coulter).Samples were % 39 analyzedusing an argon;ipn,laserwith fluorescenceexcita- 0"5 40 tion at 488 nm,',Fluoitscence emission was collected at 41 525, 575,61O and 675 nm, respectiveiy. 0.0 /a At least 10 000 cells were collected per sample. The +5 mean of the triplicate values fbr each parameter was 44 parametric -0.5 subjectedto both and non-parametricmeasures ctr3 cD4 CD8 cD56 +) tbr data analysis.Paired /-test analysiswas performed on LymphocyteAntigens 46 each of the pairs of samples to determine differences in 11 cells positive for the four lyrnphocytemarkers. In addition, Fig, 1. Mean difference between paired sarnples. 9 M.C. Diamond et al. / Cognitive Brain Research I (2001) 000-000

were found ) increases between the two samples of the The authors wish to express appreciation to the ladies in 2 drawn from each 73 blood of three women who did not play the Orinda Bridge Clubs who generouslycontributed their 2 1^ but only sat quietly bridge, listening to gentle music at the blood and bridge playing skills. This work was supported 2 time period same during which the experimental group by a University of California, Berkeley, Faculty Research 2 76 played cards.The lack change of in CD4 cell number with Grant. 2 77 this group suggested that stress, due to the procedure for 78 withdrawing of blood, was not a main factor. 7q This present preliminary experiment has indicated that References 2 80 numbers of human immune T cells, specifically CD4- 8l positive T lymphocytes, can be stimulated by someone [1] P. Bardos, D. Degenne, Y Lebranchu, K. Biziere, G. Renoux, 2 82 playing the card game of contractbridge. There is no doubt Neocortical lateraiization of NK activity in mice, Scand. J. Im- 2 83 that serious bridge playing requires higher cognitive munol. i3 (1981) 609-611. 2 P Barneoud, 84 processesassociated with the frontal areas of the cerebral [2] P.J. Neveu, S. Vitiello, P Mormede, M. Le Moal, Brain 2 neocortex immunomodulation in rats, Brain Res. 474 1ggD 394_ 85 cortex. Other mentally challenging activities could also 2 398. 2 86 bring about changesin the T lymphocyte counts. Further [3] K-F. Berman, B.P Illowsky, D.R. Weinberger, physiological 2 87 tests are needed. Our data suggest that people might be dysfunction of dorsolateral prefrontal cortex in schizophrenia IV: 2 88 able to improve their immune functions with more pur- further evidence for regional and behavioral specificity, Arch. Gen. 2 Psychiatry45 (1988) 89 poseful demanding activities related to frontal lobe tasks. 616-622. 2 t4l K.F. Berman, D.R. Weinberger, Lateralisation of cortical function 90 The correlation between the psyche and the well-being 2 during cognitive tasks: regiona.l cerebrai blood flow studies of 2 91 of the individual has been recognized since the time of normal individuals and parients with schizophrenia, J. Neurol. 92 Hippocrates,but the possible interaction between regions Neurosurg.psychiatry 53 fi990) 150_.160. 93 of the cerebral cortex and the immune system was only t5l K.F. Berman, R.F. Zec, D.R. Wqinberger, physiological dysfunction 94 brought to the attention of more recent investigators of dorsolateral prefrontal cortex in sChizophrenia: II. Role of neuroleptic treatment. arteniion- and mental effort, Arch. Gen. 2 95 through the studiesof Renoux et al. [14], Bardos et al. [l] Psychiatry43 (1986) 126-135. 2 96 and Renoux in the [15] early 1980s. They studied the [6] C. Betancur,P.J. Ngveu,rS. Vitiello, M. Le Moal. Natural killer cell 2 97 effects of lesions in the right or left neocortex on the activity is asso.giiitedwith brain euymmerry in male mice, Brain 2 98 activity of natural killer cells and antibody-dependent, Behav.Immun. 5 (t991) 162-169. 2 99 cell-mediatedcytotoxicity of splenic T cells. [7] R J. Davidson, C.C.:Coe, I. Dolski, B. Donzella, Individual differ- 2 ences in'p-reficntal aitivation asyrrrmetry predict natural killer cell 2 00 Many differencesin the responseof the immune system activity at reSt and in response to challenge, Brain Behav. Immun. 2 01 to these ablation studies have been presented and may be 93jt08. 2 02 accounted for by varying factors including extent of the i8l;"M.D. Dbvous Sr., J.D. Raese, J.H. Herman, Regional cerebral blood 2 03 lesion and period of immune analyses following the flow in schizophrenic patients at rest and during Wisconsin card sort 2 tasks, J. Cereb. Blood Flow Metab. 5 (Suppl.) (1995) 2Ol*202. 2 04 lesions. In 1999, Davidson et al. [7] demonstratedwith "'iq;,{91 M:C. Diamond, R.D. Rainbolt, R. Guzman, E.R. Greer, S. Teitel- 2 05 electrophysiological measures that activation of the prerli 14L,.''fuum, Regional cerebral cortical deficits in the immune deficient 2 06 frontal cortex influenced natural killer cell activity. Thd;' , nude mouse: a preliminary study, Exp. Neurol. 92 (1996) 311_322. 2 07 investigations clearly have demonstrated that manipuliiibn i.[0] M.C. Diamond, in: Enriching Heredity, The Free press, New york, 2 08 of a higher cognitive region, the neocorte* and more 1988. 2 G.O. Gaufo, M.C. Diamond, 09 specifically the frontal cortex, has a definite role in t11l Prolactinincreases CD4lCDg cell ratio 2 in thymus-grafted congenitally proc. 10 immune regulation. t',., athymic nude mice, Natl. Acad. 2 .=,t ,:. Sci. USA 93 (1996) 4165-4169. 2 l1 It is well known that busy, engaged p.oplqnb not t12l G.O. Gaufo, M.C. Diamond, Thymus graft reversesmorphological 2 T2 becomeill as frequentlyas idle. inactive people.positive deficits in dorsolateral frontal cortex of congenitally athymic nude 2 t3 thinking is reportedly beneficial to one's well being. Even mice, Brain Res. 756 (1997) l9l-199. 2 G.J. Hoste, t4 prayer has been implied to have a posiiive effect on one's [13] PJ. Neveu, P. Mormede, M. Le Moal, Hemispheric 2 asyrnrnetriesin the effects of cerebral cortical ablation on mitogen- 2 15 health. Are all of these stat€ments:true because an en- induced lymphoproliferation and plasma prolactin levels in female 2 16 hancedfrontal lobe protects::the individual through excita- rats, Brain Res.483 (1989) 123-129 2 t7 tion of certainimmune funciions? [14] G. Renoux, K. Biziere, M. Renoux, G.M. Guillaumin, The cerebral 2 18 Uncitedreference cortex regulates immune responses in the mouse, CR Acad. Sci. D 2 19 t8t (Paris) 290 (1980) 719-722. 2 [15] G. Renoux, The cortex regulates the immune system and the 2 activitiesof a t-cell specific immunopotentiator,Int. J. Neurosci.39 2 fi988) 177-187. 2 ).0 Acknowledgements [16] S. Rezai,N.C. Andreasen,R. Allinger, G. Cohen,V SwayzeII, D.S, 2 O'Leary, The neuropsychology of the prefrontal cortex, Arch. 2 Neurol. 50 (1993) 21 This project (#91-12-13) was granred full approval by 636-642. 2 zz the Committee for Protection of Human Subiects at the 23 University of California at Berkeley.