Single unit activity in frontal cortex and caudate nucleus of young and old rats

Neurobtology of Aging. Vol 6, pp 245-248, 1985 ©Ankho Internationallnc Printedm the U S A

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Single Unit Activity in Frontal Cortex and Caudate Nucleus of Young and Old Rats W A R R E N C S T E R N , W I L L I A M W P U G H A N D P E T E R J. M O R G A N E 3

North Carohna Foundatton f o r Mental Health Research, Raletgh, N C 27611 Burroughs Weilcome Co , Research Trtangle Park, N C 27709 and Worcester F o u n d a n o n f o r Experimental Btology, Shrewsbury, M A 01545 R e c e i v e d 31 A u g u s t 1984 STERN, W C , W W PUGH AND P J MORGANE Single untt acttvtty m frontal cortex and caudate nucleus of young and old rats NEUROBIOL AGING 6(3) 245--248, 1985 -Spontaneous neuronal activity was recorded extracellularly from ~solated single umts m frontal neocortex and caudate nucleus of young and aged F344 rats anesthenzed with urethane Average f'mng rates, mean mtersplke intervals (ISI)_+standard devmtlons, and ISI frequency histograms were computed and analyzed by m~croprocessor For frontal cortex cells (N =226), there was a nonsigntficant trend toward slower average d~scharge rates m the old group. However, a slgmficantly longer mean ISI and proportionally more very slow finng cells (<1 Hz) were observed m old rats A laminar analysis of frontal cortex umt actlvRy m young ammals showed average d~scharge rates to be distributed somewhat evenly throughout the cortical mantle wRh the exceptzon of the zone 1200--1400 t~ beneath bra,n surface Tins depth corresponds approximately to layer V where a 50% increase m mean finng rate in young ammals was observed In aged ammals, tins increased cell fmng in layer V was absent, winle mean dsscharge rates m other laminae remained essentmlly the same m the young and old rat groups Caudate nucleus cells (n=70) showed a slgmficant shift towards fewer fast discharging cells m old rats, wtth the average fmng rate dUnlmsbed by one-tinrd Although more brain regnons need to be examined m a similar fasinon, the consistency of the present results w~th those prewously reported for the bra,nstem and cerebellum suggests that slower finng rates and longer ISis are hkely to be wtde-spread throughout the brains of aged rats Aging

Spontaneous single umt acUvlty

Frontal neocortex

T H E R E ~s relatively httle descnptlve or conceptual understanding of neurophystologlcal changes which occur m the brains of aged humans and ammals Most electrophysmloglca] aglng studies m man have focused on electroencephalographic (EEG) or evoked potential (EP) recordings from neocortex by surface leads Results of EEG investigations consistently show a slowing of the mean peak frequency and increased anterior temporal focal d~scharges w~th increasing age [5,13] Stmdar age-related changes m EEG have been reported m rodents [8,27]. Although EPs are characterized by great mtenndivldual vanabflRy and senslUwty to physiological and pathological changes, the average cortical EP, especmlly the wsual EP [7,26] and the somatosensory EP [15,25] have been most widely used to measure the effects of aging Slgmficantly increased iatencles for the middle com-

Laminar analysis

Caudate nucleus

ponents and increased amphtudes of the average corucal E P m aged humans compared to young adults have been observed CollecUvely, these E E G and E P changes m aged humans may reflectd~mm~shed sensory organ functlon [l I], reduced cortlcal metabohsm [I0], progressive dendnac deterloratlon [23], and/or decreased neuronal cell numbers [4,16] One electrophys~ologdcal aspect of neuronal acawty which has received scant attenaon wRh respect to the aged mammahan brain ~s that of single umt dnscharges A study by Rogers et al [20] m the cerebellum of the aged rat found slower s~mple sp~ke d~scharge rates m Purkmje cells compared to the young adult, although bursting acuvlty by chmblng fiber sumulauon remaaned the same for the two age groups In add~taon to a marked shaft m the d~stnbut~on of

JSupported by NIA contract NOI-AG-0-219 and a grant from Burroughs Wellcome Co ~Presented in prehmmary form at the 13th Annual Meeting of the Society for Neurosclence, 1983 SRequests for repnnts should be addressed to Dr Peter J Morgane, Worcester Foundation for Experimental Btology, Shewsbury, MA

01545

245

246

STERN, P U G H A N D M O R G A N E TABLE 1 SPONTANEOUS DISCHARGECHARACTERISTICSOF SINGLE UNITS IN YOUNG AND OLD BRAINS Frontal Cortex§

No Rats No Cells Sptkes/Sec ISI (msec) Variability Ratio %Bursts¶ %Pauses#

Caudate Nucleus§

Young

Old

Young

5 105 4 1 -4 634 ± 87 1 26 ± 0 04 9± I 13 ± 1

7 121 33 ± 3 I088 ± 190" 1 28 -+ 0 05 7-+ 1

11 42 182_* 18 218 -* 74 0 66 ± 0 06 10-* 2

11 -

1

6_+

1

Old 3 28 1 2 3 ± 19" 728 ± 393 I 16 -+ 0 13~: 17 ± 3 8 ± 1

*p<0 05 tp<0 01 ~p<0 001 (2-ta~led t-tests) §Values are mean ± S E , ISI=lnterspike interval, vanabihty ratio=mean/SD of the 1SI ¶%of ISis ~<20 msec # ~ of ISis ~ twice the mean IS1 Purklnje cell discharges towards slower frequenoes, reduced excltablhty to afferent parallel fiber stimulation m old rats was also observed [21] Olpe and Stemmann [17] reported quantRaavely similar results in their recordings of spontaneous locus coeruleus actiwty m the brains of 3- vs 22-month-old rats, i e , an approximately 25% decrease m average d~scharge rates in the aged brain Spontaneous discharge rates m the hippocampus o f aged rats appear to be hkewise reduced m simple spike cells [14], although no slgndicant age-related differences were observed m hlppocampal complex cell spike height, width o r finng rates [3,14] In addition, decreased responsiveness of single cells to apphed neurotransmztters m old rats has been reported for neocortical cell responses to norepmephrlne [12] and for htppocampal cell responses to acetylchohne, G A B A and 5-HT [14,24] The present study was undertaken to assess whether the changes in spontaneous discharge rates previously observed in subcort~cai structures (cerebellum, bramstem, hippocampus) might be more pronounced in forebram areas such as frontal cortex and caudate nucleus The activity of these neurons may relate directly to some of the motor and cognitive dysfunctions that occur with old age METHOD Healthy young and old male Fisher 344 rats o f ages 5-11 and 25-31 months, were housed at 74°F on a 12/12 hr hght/dark cycle with food and water available ad hb The rats were anestheUzed with urethane (1000 mg/kg IP) and placed m a stereotaxic frame Additional anesthesm (200 mg/kg IP) was g~ven p r o re n a t a dunng the experiment sufficient to mmntam sluggish withdrawal reflexes Heart rate and body temperature were momtored continuously and remained within normal hmRs A burr hole approximately 2 mm m diameter was made through the skull above the frontal cortex or caudate nucleus recording sRe (AP=2.5-3 5 mm, M L = 1 0-2 0 mm, H = 0 - 2 0 mm or A P = 1 0 mm, M L = 3 0 mm, H = - 4 0 mm, respectively, where b r e g m a = A P 0 0) wRhout damage to the underlying neocortex Microplpettes o f 1-3 /z tip diameters were filled with 5% horseradish peroxldase (HRP) m 0 1 M phosphate buffered normal sahne (pH 7 4) and had resistances o f 5-15 Mohms Isolated spontaneous neuronal

activity was recorded extracellularly for at least 100 seconds per cell Neuronal activity was amplified (Grass P15) and displayed on a storage oscilloscope (Tektromx 5103N) Stable isolated single unit action potentials having a signal to noise ratio of at least 5 1 were dlscnminated (World P r e o sion Instruments 120) and the digitized discharge data were analyzed by a microprocessor (Apple II Plus) Mean firing rates, mean lnterspike mtervals (ISI) with standard deviations, as well as ISI histograms were calculated to provide information regarding rates and patterns o f neuronal spike discharges No attempt was made to physiologically characterize these neurons w~th respect to peripheral input Discrete marking of one or more o f the single-unit recording sites per ammal was accomplished by iontophoresing (2/~A, 2 min anodal current) a small amount of H R P from the mlcroplpette tip [19] At the conclusion of an experiment, the ammal was deeply anesthetized and perfused transcard~ally wRh 3 3% formaldehyde m 0 1 M phosphate buffer The brain was removed, blocked, placed in 10% sucrose phosphate buffer and stored overnight at 4°C 50 ~ frozen sections were reacted wRh d~ammo-benz~dine to v~sualtze the H R P marked recording sites [19] These and all other recordmg sites extrapolated from those marked by H R P were transposed to normal rat brain atlas drawings for cytoarchltectural [18] and laminar localization [6] RESULTS Spontaneous neuronal discharges were isolated and recorded from 226 frontal cortex single units and 70 caudate nucleus units m 16 young and 10 aged rats Slmdar numbers o f cells per electrode penetration per animal were recorded from both age groups Table 1 summarizes the finng characteristics of ongoing unit activity in these two nuclear areas F o r the cortex of aged rats, there was a nonsignificant trend toward slower average discharge rates However, a significantly ( p < 0 05) longer mean ISI was observed for the older rats Other parameters of cell activity, such as bursting or pauses, presented m Table 1 were comparable for young and old cortical cells The distribution of cortical cell discharge rates in Fig l (left) shows that old rats had a dispropor-

SPONTANEOUS UNIT ACTIVITY AND AGING

247

60

FRONTAL CORTEX

CAUDATE

ng

s0 E3 Young Old

[:3 Vouag ~ Old

40

40

30

3o

uJ

,~ 4o rig <

3O

//

O ~,

Old

20

10

<1

12 23 3-4 45

>5

<5 6101115162021,?,5>25

SPONTANEOUS DISCHARGE RATE (sp~kes/sec)

t 201 400

~ 4 1 600

t 601 800

I 801 1000

I 1001 1200

[ 1201 1400

I 1401 1600

1 1601 1800

I 1801 2000

DEPTH FROM SURFACE OF CORTEX (mmrons)

FIG ] Hzstogram of the dzscha.rge rates of a populauon of 226 neurons m the frontal cortex (left) and 70 neurons m the caudate nucleus (nght) of young and old rats

FIG 2 Mean d|scharge rate of frontal cortex umts In young and old rat brains as a funcuon of depth from cort|cal surface

tmnally h~gh occurrence of neurons which discharged at less than 1 spike/see (29% vs 1 5 % , p < 0 05) At spike rates above i Hz, the young and old groups were comparable Further assessment o f the cortical laminar dlstribuUon o f mean finng rates in young vs old rats m frontal cortex is shown m Fig 2 In seven of the nine recorded cortical depths taken at 200 micron increments, the mean discharge rate for the old rats was less than that o f the younger controls A zone of mcreased spontaneous sp~ke actwlty was observed m the young group at 1200-1400/z deep to the pial surface, corresponding approximately to layer V Whde this peak of faster mean firing rate in layer V was absent in the old rats, the laminar pattern analysis suggests that the aging effects shown in Table 1 were distributed somewhat evenly throughout the corUcal mantle Histological identification of HRP marked recording sites, as well as those extrapolated from the labeled sites, were examined and plotted from thmmn counterstamed sectmns This analysis revealed that unit recordings from both age groups were made in slmdar medial agranular frontal cortex regmns according to the cytoarchltectural and physiological criteria described by Donoghue and Wise [6] Although the number of caudate nucleus cells recorded (n=70) was fewer than that for the cortex, there were more significant age-related changes m cell-firing m this nucleus Figure 1 (right) illustrates the marked shift toward fewer (p<0 05) fast discharging cells m the caudate nucleus o f old rats The average finng rate was also dimimshed significantly ( p < 0 05) by one-thwd (Table 1) m the old animals The only other parameter to change was an increase in the varIablhty raho of the unit discharges m the old animals (p<0 001) This hkely reflects the presence of both decreased numbers of fasting discharging cells plus a greater population of more slowly firing, irregularly discharging neurons m the caudate nucleus of older rats

portion of cells with very slow fhang rates It should be noted that differences m mean finng rates of frontal cortex cells in our study of young and old rats faded to reach statistical sigmficance, although the magnitude o f change was slmdar to other studies in different brain structures Other parameters of cell finng, such as mean mtersplke intervals and percent slow firing cells (<1 Hz), as well as mean spontaneous finng rates for caudate units, were statistically different between the two age groups While an age/anesthesia interaction cannot be ruled out absolutely, the magmtude of the present findings are comparable to those reported using different anestheucs for the brainstem [ 17] and the cerebellum [20] in the aged rat, 1 e , a 20-30% decrease m mean discharge rates Moreover, luppocampal umts m the rat studies by Llppa et al [14] using chloral hydrate anesthesia revealed a similar 25% overall decrease In firing rates Upon further analysis, they found that s~mple spike cells slowed 40% in aged rats but no changes m the firing rates of complex spike cells were noted This observation has been confirmed m the unanesthet~zed, freely-moving rat by Barnes et al [3], who likewise found no differences m hippocampal complex spike height, width, or firing rates between young and aged rats Relatwely smaller age-related changes in neuronal reactlwty were noted m the response of cells to iontophoretlcally applied transmitters when compared to the marked anatomical changes of aging (cell numbers, synaptlc density, etc ) These small changes observed in response to iontophoretlc stimulation may be due to compensatory receptor mechanisms In the case of the hippocampus, aging is correlated with a reduction in the number of synapses [9], but a concurrent increase m the efficiency of synapt~c transmission [1,2] S~mdarly, changes m transmmtter synthesis, release and re-uptake may be offset or compensated by a change In their physiological action in the aged bram In our cortical laminar analysis, the greatest age differences were found at the 1200-1400 V. depth where average f'uang rates in young but not old rats were increased by 50% This corresponds to the large pyramidal cell layer V, the same region where decreased d e n d n u c arborizatmn and synaptic density deficits have been described m aged hu-

DISCUSSION

The present results from frontal cortex and caudate nucleus show that age-related changes occur m spontaneous neuronal discharge characteristics m the rat braun Observed effects were reduced average spike activity and a higher pro-

248

STERN, PUGH AND MORGANE

mans by Schelbel et al [23] F u r t h e r m o r e , this is also the s a m e cortical lamina (V) w h e r e ascending cholmerglc basal forebram afferents terminate [22] A l t h o u g h m o r e brain r e g m n s need to be e x a m i n e d using the same t e c h m q u e s , similar patterns o f s l o w e r and m o r e irregular unit discharges o b s e r v e d in the p r e s e n t report and t h o s e studms p r e v i o u s l y d e s c r i b e d suggest that these changes are p r e s e n t m several areas o f the brains o f aged

rats It ts equally interesting to note that s o m e brmn structures, such as c o m p l e x spike cells in luppocampus, do not s h o w s l o w e r s p o n t a n e o u s finng rates w~th increasing age W h e t h e r these age-related changes reflect d e c r e a s e d cerebral metabolism, selective cell death, reduced excitatory input, diminished post-synaptm responstvity, s o m e other m e c h a n i s m , or a combination o f the a b o v e , remains to be determined

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14 Ltppa, A S , D J Cntchett, F El-alert, H I Yamamura, S J Enna and R T Bartus Age-related alterations in neurotransminer receptors An electrophysmioglcal and biochemical analyszs Neurobml Aging 2: 3--8, 1981 15 Luders, H The effects of agqng on the wave form of the somatosensory cortical evoked potential EEG Chn Neurophvs1ol 29. 450-460, 1970 16 Mountjoy, C Q , M Roth, N J R Evans and H M Evans Cortical neuronal counts m normal elderly controls and demented patients, Neurobtol Aging 4 1-11, 1983 17 Olpe, H R and M W Stemmann Age-related dechne m the activity of noradrenerglc neurons m the rat locus coeruleus Brmn Res 251: 174-176, 1982 18 Paxmos, G and C Watson The Rat Brain m Stereotax~t Coordinates New York Academic Press, 1982 19 laugh, W W and W S Stern Horseradish perox~dase labehng of extraceilular single unit recording s~tes Brain Res Bull 12" 419-423, 1984 20 Rogers, J , M A Silver, J Shoemaker and F E Bloom Senescent changes In a neuroblologlcal model system Cerebellar Purlonje cell electrophyslology and correlative anatomy NeurobmlAgmg 1 121-124, 1980 21 Rogers, J , S F Zornetzer and F E Bloom Senescent pathology of cerebellum Purkmje neurons and their parallel fiber afferents Neurobtol Aging 2: 15-26, 1981 22 Saper, C B Organization of cerebral afferent systems in the rat I Magnocellular basal nucleus J Comp Neurol 222 313342, 1984 23 Scheibel, M E , R D Lmdsay, U Tomlyasu and A B ScheJbel Progressive dendnt~c changes in aging human cortex Exp Neurol 47: 392-403, 1975 24 Segal, M Changes in neurotransmmer actions in the aged rat hlppocampus Neurob:ol Aging 3. 121-124, 1982 25 Shagass, C and M Schwartz Age, personahty, and somatosensory cerebral evoked responses Scteme 148. 13591361, 1965 26 Straumanms, J J , C Shagass and M Schwartz Visually evoked cerebral response changes assocmted with chromc brain syndrome and aging J Gerontol 20 498-506, 1965 27 Zepehn, H , W E WMtehead and A Rechtschaffen Aging and sleep in the albino rat Behav Bml 7" 65-74, 1972