Alterations in intracellular calcium chelation reproduce developmental differences in repetitive firing and afterhyperpolarizations in rat neocortical neurons

DevelopmentalEnin Rescarcb84 (1995)192-203

Research

report

Alterations in intracellular calcium chelation reproduce developmental differences in repetitive firing and afterhyperpolarizations in rat neocortical neurons N.M. Lorenzon, R.C. Foehring acprirmrmr ofAnatomy andNnvobidoey.

855 M-

Avenue, IJhersity

??

of Tatneswe

- Men@&

hfmrphir. TN Mi63, USA

Accepted 20 September 1994

Many’l-week-old rat scnsorimotor cortical neurons cxhiiit extreme spike-frequency adaptation (neurons only fire for the tint ?!I@-2% ms of a 1 s current injection) a-penicd hy a large, prolonged nfterhytterPolarlzation @HP). Relatively sreater expressionof a Cadependent K+ currentappearsto underliethe extremeadaptationobservedin immaturecells. In the present study, we examinedwhether altering intracellular Ca*+ buffering by introducing Ca*+ chelators via the recording electrode could reproduce the age-related differences io firing and AIWs. WC studied firing behavior and AHF’s in l-week-old and adult neam&al neurons with sharp microelcctmde~ under three recording conditions: no chelator, 2 n&f BAITA, or 100-290mM BAPI’A.Our principal findings in regard to firing behavior and AHPs were that (1) adult-low BAFTA neurons mimicked 1 week-control cells, (2) 1 week-high BAPTA neurons were similar to adultcontrol cells, (3) a greater percentage of 1 week-low BA?TA neumns shmved complete adaptation, and (4) adult neurons impaled with high BAWA electrodes fired in a burst-spiking mode. These data suggest that Ca*+ regulation is qualitatively diierent in immature and adult neurons. &WV&:

Fhing behavior;Afterhyperpolarizati;

Development; Cortex; Ca~ependent

1.Intmdactien We re.centIyreported that tbe integrative abilities of neocotticaI pyramidal cells undergo a marked change over the first month of postnatal development 191.In adult neurons, the firing behavior of tite majority of ncocottkzalpyramidal neurons is characterixcd by tonic, repetitive spiking and modest spike-frequency adaptation (decrease in instantaneous firing frequency with time) during a long, depolarixmg current injection. The average firing frequency increases in adult neurons as the stimulus intensity is increased. In contrast, many immature neurons respond to long current injections with a more phasic mode of firing; they cease firing after the first 100-250 ms regardless of stimulus intensity. In adult cortic=aIneurons, spike-frequency adaptation is primarily due to &dependent K+ conduc-

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compoadi;i6 ai;thor.Fpx:(I) (901)448-716.

0165-3&%/95/$09.50Q 1995ELmicr ScicoccB.V. All d&s resewed SSDI 0165~3806(94)00171-5

K+ current; BAPTA

tances which are also the main contributors to the subsequent slow afterhyperpolarixation CsAHP) [1,7, 11,221.A greater relative expression of the same Cadependent K+ currents appears to underlie the extreme adaptation seen in immature ccUs. This is sup ported by the observation that nemotransmitters (or inorganic ca ** blockers) known to reduce these currents in the adult also reduce adaptation in immature cells. These agents allow the cells to Rre throughout the stimulus in a pattern more similar to that of the adult [9]. Several mechanisms could account for the age-dependent differences in firing behavior, including developmental diierenccs in (1) Ca-dependent K+ conductanccs (e.g. greater density, slower channel kinetics or increased channel sensitivity to Ca’+ in immature cells), (2) voltage-gated Ca*+ currents (which could lead to greater Ca*’ availability: i.e. greater density of channels in yomtg animals, changes in voltage-dependence or kinetics, or less C&dependent inactivation of Ca’+ currents in immature animals), or (3) Ca*+ regulation

N.M.

Loreruon.

RC.

Foeh,,,,g,DmclopmenraI

(levels or types of Ca*+ buffering proteins, release or sequestration of Ca” from internal stores, Ca2+ entry or extrusion via a Na+/Ca” exchange system). A recent study by Schwindt and colleagues [2!] suggested that different levels of CaZf chelation can lead to changes in tiring behavior and AHPs. In that

1,2-dis (o-aminophenoxy) ethaie-N,N,N’, N’-tetraacetic acid (BAITA). Cat Betz cells, like rat sensorimotor cells, have Ca-dependent K+ conductances which under$e their slow AHPs and spike-frequency adaptation expected, at high concentrations (100-200 mM), BAPTA reduced the Ca-dependent K+ currents. H current and the persistent Na+ current were also redufrd *‘. At low concentrations (2-20

. As

impaled with electrodes filled with 2-20 mM BAPTA appeared similar to that of the immature rat neocortical cells that we have previously described [9], we tested whether altering Ca2+ buffering in rat neocortical neurons (by introducing BAFTA via the recording elect&c) could reproduce the diffcrenccs ir! firing and AHPs observed between the adult and immature cells. That is, can altering the Ca2+ buffering ability of the cell convert a mature neuron into one that behaves like an immature cell and/or an immature neuron into one that behaves like an adult cell? Successful mimicry of age-dependent differences by &eiatioll would suggest that differences in Ca2+ entry or Ca” regulation underlie age-dependent changes in tiring and AHPs, rather than changes in numbers of Ca-dependent K’ Lhannels.

2. Materials

and methods

Necartical “eumns were studied using a” in vitro slice preparation. Adult ( > 6 weeks of age) and munature(P6-33) Sprague-DawIcy rats were anesthetized with methoxy-fluorane (Metofane) and sacrificed by decapitation while under anesthesia. [Most immature cells were hum l-week-old animals G’6-IO)]. A b&k of tissue was removedfrom the senwrimotor region of anterux cortex.The msuc sample was immediately sectioned (400-500 pm thick) in “omtal artifiiial fcrebrospinal fluid (&SF) at 4T using a WF’I oscillaling tissueslicer (For l-week-old animals, 500 PM sections were used: we found this inctased cell viability). The &SF contarncd (in mM): 125 NaCI. 3 KCI. 2 C&I,. 2 U&l,. 1.2 NaH,PO,. 26 NaHCO,. 20 dextnse fpH 7.4: 310-320 mOsm/L). Sliceswere then incubated for at least I h at 3lPC in a holding chamber containing normal &SF bubbled with carbogen (95% 0,. 5% CO,). Individual sliceswere then transferred to an interface recording chamber maintained al 34’C. The slii was bathed in oxygenatedaCSF which flowed under the slii. Carbogen-saturatedwa(er vapor flowed over the lop of the slice. To observe the effects of zero Ca’+ on cells, the Ca*+ in the &SF was replacedwith 2 mM Co2+. and Ihe NaH2F’OI was omitted to avoid precipitation. The B_adrenergicagonist iwpmtcnnol (HCI;

Eram

Research 84 (1995)

192-203

SO-100 p iri; obtamed t-tom S&a Cbemiad Co.) was added dire& to the aCSi= lust prim to appficatiat to the cell. Saditmt wtrbisultite (50 pM) was F&o added to the bath solution to reduce the oxidation of isoprotmad I241 Contrui rmcrcekcnodcs MO-180 MJZ) were tilkd with 2 M KMeSO, or 1% biwyti”/;! M KM&O, s&ttiotw BAFTA ckcfilkd with L&4FTA 12 01 100-200 mM: trades (75-16.5 MR) Calbiochem) dissolved in 2 M KM&O, (ii &&ti”n. 5 adtdt cella were impaled mth 2 mM BAPTA d&s&cd i” 3 M KCI: I)Odifferencn were found betwee” the KCI md K&SO, ek.ztrak rcrmd mgr I” the adult celk, so these data were pctded). I” add&m, sawal electrodes wte lilkd with EGTA (2 mhl or MO-2IMlmM; Caltuochem) dwlvcd in 2 M KM&O,. Rccordiirg were from cells ,” layers II-V (wth “test in *yen 11/111 (K v). All recordings utilized a” Axo&mp-IIb ekctraneter (Axoa Iastrttme”t.5) I” m”tintmtt.9 btidge ot disamtinttous cum”t clamp (DCC)mode.InDCCmode.r2_4Hr~ntcand3096duy

obtamed The bead-stage “utplt was viewed OD a separate G5c~llosc0pe. a”d pub&bed ptwedwsI2IRlefouwcdtoset capacttance compemptiott conxtly a”d to tmu&izc *=ttMt% rate Data were r& and sto,ed oa videoupc using a ti corder (Newdata) and a VCR. AHF% and firing frequencywere. wualued and measured off-lint with RC Electma& caapltcrscope Rcsung membranepotenlisk were estimatedhum amtinuow chan recordmgsof DC polential. The cells were dii ittto 6 /toups by ale a”d BAFTA axtce”. trano”: weekcontrol (no MA), 1 week-b* BAFTA (2 “MI, 1 week-high BAFTA (MB-200 tt,M). adtdtswtrd, adult-tow BAPTA, and adult-h& BAF’TA AU statistical data are presented as mean+ SD Sta!!z!!cz! dlffere”cS between vaiotts groups were detctmiacd by a nonparametric test (Kntskal-Wallis ate-way aarlrs;s of vatiaxe/Mann-W’hanq U-test), or Fir’s unct test (x2 an&&l. SGTZ nf :!I.- dzra for anrtml neurons fxeseated in this paper were dewad from (91.

cycle were

I

3. Results 3.1. Normnl

b&CdO~

in

oakit and

I-wedc-okd

The following data were obtained from successful intracellular impalements of adult (> 6 weeks of age: n = 167 cells) and immature (P6-33; 228 cells) neurons. All cells had action potential amplitudes of > 60 mV and input resistances of > 20 MS). Adult rat regular-spiking neocorticai neurons [1,12] fired in a regular and pattern in response to a 1 s depoiarizing stimuius pulse, and exhibited modest spikefrequency adaptation (Fig. 1A). At 1 week of age (P6-10). 60% of neocortical cells (U/40) exhibited a phasic pattern of firing @ii. 1D). We defined M#IIpletely-adapting cells as ~urons in which fuing terminated within the first 100450 ms of the stimulus pulse regardless of stimulus intensity [9]. The remaining 40% (17/40 cells) of the 1 week old ceils fired regularly throughout the stimulus (Fig. 1D. inset; see [9D. (Cells that fired only one spike, took > 1.0 nA stimulus current to generate an AP, and lacked medium or slow AHPs were considered injured and were not included in these percentages. We have previous& shown by

tonic

NM Lnmum, RC. .%chrhg/Kkuclopnm~d bin

&search 84 (1995) 192403

1WEEKm

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QmV

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L

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L

Fii 1. Repetitive firing of adult and I-wek-old nwcorticrl neurons with Kh%SG,- and BAPTMilkd elecInxk~. A: repetitive tiring from an whdt ncumn imp&d with P KMeSG4 ek%Irak (0.5 nA for 13 Note the tonic repetitive tiring panem. Gains for A-F are as noted in E. 8: rcptitivc tiring fmm a F8 neuron impaled with a 100 mhl BAPTA ckctmdc. (0.75 nA for 13 The tonic firing paItem is similar to that of the adultsontml cell. C firing from adult all imp&d with a 100 mIb4BAPTA ekchc&. This all fired in #mups of bun& tbrc&out the stimulus pibe (05 I&. The fint burst of spiker (marked by asterisk) is shown at faster sweep speed in the inset. Note different pins for inset. Note alsn tke much reduced sAHP. D: Rring of a com~tely-adapting (10098 adaptation by 250 ms) 1 mckcoaIrol neuron. Re~kss of stimulus intensity, UK neuron would nol fire thmu#hout a 1-s current injection. 60% of cells tested at 1 week were mmplctely-adapting. Note large sAHP. IN*: the remaining 40% of the cells lkcd thmu&out the current injcctkm (repetitively-fixin cells). Gains fm inset arc as noted. E: firing bchavfor of a I-mek-old ncumn imp&d with I 2 mM BAPTA ekctmde (1.0 nA for 1 a). NOICcomplete adaptation and lame sAlif’. F: finap fmm an adult neuron imo&d with P 2 mM BAF’TA tbtimde (1.0 nA for 1 s). Note Ike extreme soike-freaueaev adaatation and lane SAM’.

several criteria [91 that the completely-adapting firing pattern in some immature cells does not reflect injury. These criteria include action potential amplitude, resting membrane potential, input resistance, the presence of a large, prolonged sAHP, and the fact that these cells wig fiie repetitively in the presence of NE or inorganic Ca*+ channtl blockers.) Percent spike-frequency adaptation was defied as

[(average firing frequency over the first 200 ms)(average firing frequency over the last too ms)/average firing frequency over the first m ms; all cells were compared using a 0.5 nA, 1 s current injection as the stimulus] 191.The 0.5 nA current was arbitrarily chosen as an intensity which could be achieved in all cells and induced firing in the primary slope range in adult cells [9]. For l-week-old ceils (all cells pooled: completeiy-

Tabkl Bffects of BAPTA on repctitiw tiring and AfiP propertics of ncocorIical neurons Bkctrical properties

Adult control

i W-k LawBAPTA

HiahBAPTA

Controt

Hinh BAFTA

3.2. AHPs in l-week-old

and adult neurons

Following a train of perpolarizations were medium AHP (mAHP, larization immediately

action potentials, two afterhypresent in adult neurons: a measured as the peak hyperpofollowing the stimulus) and a

A mmv

ADlJLl.coNTRoL

slow AHP CsAHP; measured at 500 ms after the cessation of the stimulus pulse) [9] (Fig. 2A; see also [1,8,22JJ. In l-week-old cells, both medium and slow AHPs were observed. The extreme spike-frequency adaptation of completely-adapting cells was accompanied by a large, prolonged =A_YP (Fig. 2D). To allow more direct and quantitative comparisons, AHPs were elicited by trams of action potentials contaming a controlled number of spikes. Fig. 2 shows AHPs evoked by 2-20 suprathreshold, tms current injections delivered at 100 Hz. The amplitude of the mAHP did n>t differ significantly between adult and 1 week-control neurons. This was true whether the mAHP was measured following 20 spikes (Table 1) or a single spike (data not shown). The sAHP ampiitnde foliowmg 20 spikes (5.9 f 4.1 mV, n = 24) was sign& candy greater in the l-week-old cells -pared to tbe adults (2.0 2 1.7 mV, n = 8; Table 1). The AHP duration was significantly longer in l-week-old cells than in adult-control cells (Table 1). Even greater differences in AHPs were seen between adult and immature cells after small numbers of spikes (see below); Fig. 3 graphically deprcts AHP amplitudes and durations after 5 spikes (elicited at 100 Hz). In summary, at 1 week after birth, neocorticaf neurons exhibu one of two firing behaviors: completelyadapting or repetitively-firing. The firing behavior of l-week-old, repetitively-fling cells appears similar to

1 WEEK OLC4GH EAf’TA

AOULTSWOHBAPTA

5,10,303PlltE9 “,*

.z

428-

Fig. 2. AHR of adult and I-week-old neoconical neurons with different mncentratmns of BAPTA in the ekcttwks. A: AHPs from an adult neuron impaled with a KhieSO, electrode.The AHR were elicited by repetitive.suprathres!tdd 2 ms stimuluspadsesat IO0 Hz; 5, 10.30 spikes (preceding spikesnot shown in A-F). Note the small sAHP; 10 spikesor more am needed to elicit a mes.;urahk sAHf’ In A-F. the medium AHP is indicated by filled circle and the slow AHR by empty squares. Gains for A-F dlustnted in E. B: AHPs eliiited fmm a PJ3tteuron impaled with a 100 mM BAPTA ekctmde (5. 20 spikes).Note the simdaritlesto the adult AHPs (the sAHP is even smalkr in this cell than in ttdult-amtrol cells). C: AHPs from an adult cell impaled with a 200 mb4 BAFTA electrode. Note the lack of a sAHP. AHF’s cliiited by 5, 20 spikes. D: AHR in P 1 wek-eontrol neuron (2.5.20 spikes). Note: the sue of the sAHP. only 2 spikesare mquired to cIiit a sAHP. note aho the kinetic separatmon of the mAHP and prolongedsAHP (especially&dent at low spike numben). E: AHPs hwn a 1 week-lowBAPl’A cell 6, IO, 20 spikes) F: AH!% from an adult-low BAPTA cell (AHPs elicited by 5. 10. IS. 20 spokes).Note the similarity to I wek.cotUml neutws in the size of the sAHP, the small number of spikesrequired to elicit a sAHP, and the kmet~cseparation of the medium and shw AHPs.

N.M. Lonnrm.

RC. Foehring/Lkc&mental

Bmin Rsemrh 84 (1995) 192-203

that of adult regular-spiking neurons. One-week-old, completely-adapting ent tiring pattern 3.3. Lomirwr

cells fire phasically; than adult cells.

dim&&on

of I-week-old

a very differ-

et&

Completely-adapting cells were not restricted with

B

7

T

T

-r

Fig. 3. Frequency histogramsfor perceut adaptation, sAHP amp& huJc, and AHP duration (data pookd for compktely-adapting and naIitiv&-fifinr cells). A: diffefcnces in ~emnt (5%)adaoMon new&s with c&ol, l&v BAPTA,~and hi& BAPTA clectraks. Percent spike-frequencyadaptaticm w.s defined as the following rektiihip from a 0.5 nh I s current b+tii: [(average firing freQwncy over the fmt 200 msMavengc filing hzqucncy of the last 200 ms)/averpse ftig frequency over the first 200 msl. Absolute valuea can be found in Tabk 1. B: differences in average AHP amplitudes al3er5 spikes elicited with 2 ms reptitivc rtimuli at a fnxuency of 100 Hz. The amplitude of the sAHP was measured a 500 ma after stimulus ccssatian. sAHP amplitudes (in mV): adultcontrol, 0.6tO.8 (n - 3); immature-biih BAFT& 0.6kO.6 (n - 5); adub-hi& BAPTA, 0.8f0.4 (n - 3); imnuture-amtml. 4.3f3.5 (n - 7): immature-law BAPTA. 3.Ok2.0 (n - 7h adult-low BAPTA, 2.9k3.6 (n = 5). C: Differences in avcr~c AHP durations after 5 @es Cwmc protcwl as in 9). AHP duration fin sccwdsJ):adultc~atml, 0.2f0.2 in - 3); inuna~ure-bi BAPTA, 0.2*!?1 (n = 5): xlult-high BAPTA, 0.3fO.l (n - 3); immaturecwtrol, 3.6+ 1.9 (n _ 6b immalu~&~ BAI’TA, 3.6i: 1.7 01 - 7), adult-low BAFTA, 21 f 15 (n - 5). Symbdr: (#) denotesparameters hum the 1 weekWWOI noup that ue signifftly different from aduit-control alis, and (* ) indiated parameters sigdliiUy affected by BAPTA impskmesij as compared to control cells in the sameogc group.

respect to cortical layer. They were found in layers I1 through V at all ages in which they were enccuc:c:cd (P6-P28). The frequency with which completely-adapting ceils were observed did diier between cortical layers, however. When all layers and ages (P6-28) were analyzed together, 42% of the cells (50/119) were completely-adapting vs. 58% repetitively-firing (79/119 cells). For P6-28 combined, layer III neurons (64% completely-adapting: 21/33 cells) were significantly more likely to be completely-adapting than layer V cells (34% completely-adapting: 29/86 cells; P = 0.002 that these proportions would be obtained by chance; Fischer’s exact test; n = 119 cells). Superficial and deep cortical layers are known to differ temporally in their maturation [Z&14,17,271. In 1-weekaId animals, 61% of the cells were completelyadapting (23/38). Seventy-nine percent 09%) of layer III cells (U/14) were of the completely-adapting type at 1 week, compared to 50% of layer V cells (12/24). In 3-weekqld neurons, 33% of layer V c&s (5/15) and 33% of layer III ceils (l/3) were completely-adapting. Thus, completely-adapting ceils were ez~~un~ered more frequently at the youngest ages, and at those ages more completely-adapting cells were found in layer III than layer V. Given the Inside-out developmental sequence of cortex [25], these findings suggest that complete adaptation is an immature firing pattern. 3.4. Effects of intracellular

BAPTA on action potentials

have previously described differences in passive and active eltctrical properties between l-week-old and adult neocortical neurons (91 (see also [6,13]). We

Consistent with our previous study, we found that resting membrane potential and rheobase did not differ between the 2 age groups (Table 2). AP amplitude, spike width, dV/dt for both the upstroke and down-

stroke of the spike, and input resistance all differed significantly between l-week-old and adult neurons (Table 2) [9]. Within each age group, BAPTA had some effects on passive membrane properties or action potential wave forms (Table 2). In adult cells, low BAPTA significantly increased input resistance and reduced the rate of action potential depolarization; high BAPTA signifi-

cantly increased spike width and reduced the rate of depolarization, In l-week-old cel!o, !ow and high BAPTA both significantly reduced the rate of action potential

repolarixation.

Regarding

our initial bypothe-

ses, impaling adult neurons with !olv BAPTA e!erand trodes did not produce cells with AP amplitudes spike widths similar to those of the 1 week-control cells. Additionally, impaling immature cells with BAPTA electrodes did not produce action potential or passive electrical parameters similar to that of adultcontrol neurons.

fired m hurstf of action potentials Wig. 1C: 5/10 cells fired in a bursting pattern, 3/10 cells were reguhuspiking, Z/IO ceils wouid not fire more than 1 spike). The +king pattern of the burst-firing cells was similar to that of the small pq~lation of b&n&ally burst-f%ing neocortical cells found in deep layer IV and layer V [:,121. The high BAPTA cells exhibiting this bursting behavior were, however, not restricted to layers N/V (3 cells were found in layer 111 and 2 cells in layer V). Spike frequency adaptation was noH)tquantified in these cells since the cells fired in groups of spikes; average finng frequency was not a meaningful statistic (Fig. 1C; T..l.IL0V.C 0 .I. Fifty percent (6/12) of immature neurons impaled with 100 mM BAl’TA electrodes would only fire 1 spike at the beginning of the stimulus pulse. This mablhty to fire repetitively may be due to injury or related to a reduction in a persisten: Na+ current (cf. [21]), or other K+ currents. The remaining 6 cells all fired repetitively throughout the stimulus and exiiited modest spike-frequency adaptation (44%; more similar to that I>f adult-controi cells; Fig. lB, Table 1). Percent adaptation was signifiiantly reduced in 1 week-high BAPTA cells compared to the 1 week-control group, but did no, differ significantly from ad&control cells (Fig. 3X; Table 1). At neither age C:-week-o:d or adult)

3.5. Effects of intracellular BAPTA on repeiitlce firing and AHPs In general, neurons impaled with BAPTA electrodes exhibited quantitative and qualiiative diffcrences in repetitive firing and AHPs, compared to cells impaled with normal electrode solutions (no B.UTA). These differences were dependent on BAPTA concentration and the age of the animal. After a recording was stabilized (negative d.c. current was applied durir.g stabilization), the effects of the chelator were generally apparent immediately (in a few cells the BAPTA effect became more dramatic with time after impalement). J3ecause of the rlpidlty of the BAITA effect, conilul traces geneml!y could not be obtained before the chelator took effect. During a given experiment, we routinely alternated between recordings with KMeSO, and both high and low BAPTA electrodes, to ensure that effects were not dominated by inter-animal or -experiment variability. In addition, experiments on i~~a>~rc and adult cc!!s ivere aiZrcz:c!;. performed over ahe same time period to control for variations in technique as opposed to age or treatment. 3.6. Effects of BAPTA on repmwe

were any completely-adapting BAPTA

Adult

ceils observed with high

electrodes.

impaled with 100 mM EGTA eleefiring behavior similar to that of cells; they were burst-firing and had greatly reduced AHPs (n = 4; data not shown).

trodes

neurons exhibited

adult-high

BAFTA

firmg 3.6.2. !JJOW BAPTA Impalements with low BAPTA electrodes produced different effects on firing than those with high BAPTA electrodes. In the adult-control group, complete adap tation was necer observed. Mean percent adaptation in

3.6.1. High BAFTA High BAPTA (100-200 mM) had varying effects on the firing behavior of adult cells. After being impaled with high BAPTA electrodes, many adult ccrtical cells

Table 2 Effects of BAYTA on electrical pmpenws of newort:cal neumns 1 Week

Electrical properties Adult Contml Rcstinp potential (mV) AP amplitude (mV) Spike width (ms) dV/dt. up W/s, dV/dr. dvwn W/S) Rheobase(~4) lnpt resistance (ML?)

-7r:

*

7(57)

91 * ll(103) 0.8 * 0.2 (100) 339 f 88(89) 73 Yt39(89) 0.4 * 0.4 (93) 42 f2EM4)

Low BAPTA

High BAPTA

COlltrOl

-60

-68

-67

f

6W

94 * 15 (48) 0.8 * 0.2 (48) 245 f70W)’ 74 + 2lwO) 0.3 * 0.2 (31) 5’) f 33(28)

f

6(143

88 * IJ(iZ) 0.9-t 03(1?). 243 i76Cll). 54 * 19(11) 0.4 f 0.3 (6) l8(6)

* 40f

76

i

6(W)

*

9f47)’ 0.9M.4) ’

?.I*

140

*86(36)

LOWBAFTA

Hi& RAFTA

-68

-68

78 f 2.4 f 86

44 *L8(36)’ O3f 0.306) M9 i 466(1Y)’

f

31cl) E(6) 0.4 (6)

f U(6)

26 f 12 (6) . 0.4 * 0.4 (5) I15 *72s

f

Si13)

74 * E(12) 2.3f 0.3(12) 89 f3ZflO) 24 f 6UO). 05 f O.S(6) 62 f34C.n

Values expresxd as mean f SD. (number of cells). Spike wdth measuredat half amphtude. dV/df up is the rate of rise of the action ~~:tnt*l, and dV/dt down is the rate of rcpolarization of the spike. RbeobascIS the mimmumamount of curret to elicit an action potcnt*l tith 2fJfJ1111 cwrent m,ecuon. (‘) marks the I-week-old control paramefen that were swuficantly d,ffereat front ad& mntro! cc%. ?..: ZiStCriSk (‘1 denOti valuesthat were significantlydifferent from the controls of the same age group Kruskal-Wallis similar resullsobtained with 1-M). Some of the data for adult (7/25 c&s) and I week (S/33 cells) control ncumns were derwed from t91

test:

N.M. Lenmum, RC. Fa+ring/L2tx&mmatBmin

adult-control ceils was 38%, and the median was 34% (n = 25). Impaimg adult ceils with low BAPTA eiec@odes increased spike-frequency adaptation dramaticaiiy (Figs. 1F and 3Al. Twenty-three adult-low BAPTA cciis (out of 41 cciis) were tested for percent adaptation (0.5 nA, 1 s current injection). The mean percent adaptation in ail adult-low BAFTA ceiis combined

Reswreh 84 (1995) 192-m3

(87%; median = 100%: n = 23) was significantly greater than those of the adult-control group, and was similar to that of the 1 week-control group (Fig. 3A; Table 1). The adult-low BAFTA ceiis which fired throughout the stimulus pulse (repetitively-tiring) had significantly enhanced adaptation (79 f 24%; n = 141 compared to aduit-controi ceiis (only 2 adult-low BAPTA ceils had percent adaptation less than 73%, which is considerably higher than seen in adult-control cells). When complete adaptation is defined as cells that show 100% spike-frequency adaptation rcgardlcssof stimulus intensity, 54% of adult-low BAFTA ceiis W/41) were ciass&d as compieteiy-adapting. Thus, altering Ca*+ chelation (by Introducing low concentrations of BAETA in the recording electrodei induced a more immature firing behavior in adult neurons. In 54% of adult-low of the firing pattern of RAPTA ceiis, this mimii cells was complete. No immature comyleteiy-adapting correlation existed between the probability of an adultlow BAPTA ceii exhibiting complete adaptation and

-l-w

*IIclmw

Pi& 4. The differencea in A&s end repetitive firin@in immature and bw BAPTA calls were not due to differences in spike width or input rerirtmcc. To control for ye-sad BAITA-r&ted diiennctr in spikewidth,we integratedbver time) the areaunderthe action pocea~uredtoclieitAHR.S~~~~&~fmmlweek&l ceiir (empty cireks are repetitively tlrinc cells and filled circka ale campktely-&d8ptin~ails), and aquarea are data fmm in adult ne~mm, the duration of the sAHP incnxwd with an increase in the area under the spikes. 8: l-week.& cells imp&d with hi BAPTA electrodeskhavcd CmiMy to adult-control neurons, where wr AHP duntionr arc seen at lamer spits areas. C: in the adult-highBAPTA cells wry littk or no &HP could be elicited. D: in l-week-old neurons, the AHP duration and ma appead to have a quslitstiveiy different relstionship than that in the dult CA).Repctiriwty-firins cells from l-week-old raw &wed DOrelationship between AHP duration and spike area. In contrast, cawktelwdwthm cells showed an inverse relatiolubb with the b&t AilPa &t&d hy smalkr spike asas. E: l-&k-old celb imp&d with tow BAPTA ckctmdes resembkd immature canpktclwdaptin8 amhul cclk R adult-lw BAITA alb exhibited a AliP-inte#rated area rcMiomkipnimiiuto thst d 1.week&d ccapktely-ad@o# celb. Note: even though BAPTA dii not broaden spikes or chula inplt resistance *t bw L7Jncen~tklns.the immatwe-like t%in#and AHPs were still atqarent. Sow of the data for adult- and 1 week-controlcells werz dciived from [9L

adult CCL. A:

the lamlnar location of that ceil (P = 0.54, x2 analysis). Eighty-eight percent (7/8 cellsi of 1 week-low BAPTA ceiis were completely-adapting, compared to 60% of 1 week-control cei!s (Fig. I!3 Two of these ceils were from layer III, and six from iayer V. The mean percent adaptation was similar in 1 week-low BAPTA ceils and 1 week-control ceils (Fig. 3& Table 1). One wouid expect that, if calcium buffering underlies the diierence between repetitively-firing and compieteiy-adapting ceiis, some repetitively-firing l-weekold ceils wouid be converted to the compieteiy-adapthrg type by low BABTA electrodes. In 1 week-control cells, we found :hat 48% of layer V cells (11/U) were completely-adapting. Eighty-three percent (83%) of the layer V cells (S/6) impaled with iow BAPTA eiectmdes were completely-adapting, suggesting that some l-week-old layer V ceiis which previousiy were of the repetitively-firing type were converted to completeiyadapting cells by low BAFTA. This could explain the higher percentage of completely-adapting ceils in the 1 week-low BAPTA group compared to the 1 week-control group, although we can not rule out effects of sampling variation and small samples. Moat adult ceils impaled with 2 mM

EGTA eiectrodes (7/8 cells) exhibited complete adaptation simiiar to adult-low BAFTA ceils (data not shown). 3.7. Effects ofintmcellular BAPTA on AHPs 3.7.1. Hi& RAFTA High BAHIA impalements of adult ceils did not signifhxntiy alter the amplitude of the mAHP (after either a singie spilre or repetitive activation) compared Co adult-control ceiis (Table 1). The sAIIBs in aduithigh BAFTA ceiis appear reduced or nonexistent compared to aduit-control ceiis Wgs. 2C and 3; Table 1).

N.M. Lmenm, In 1 week-high

BAPTA

RC. Foehting / ?3cMopmenmal&am KcKorch (ij 11995) 192-203

cells, the amplitudes

of the

medium and slow AHPs after 20 spikes (2 ms pulses at 100 Hz) were significantly reduced compared to 1 week-control cells (Fig. 2; Table 1). AHP duration was also significantly reduced in 1 week-high BAPTA cells, compared to 1 week-control cells (Fig. 3C. Table 2). One-week-old neurons impaled with high BAF’TA electrodes exhibited sAHPs more similar to the aduitcontrol cells than to those of the 1 week-control cells (Fig. 2B).

larger n = 9).

t!xxi in

i99

(2Oi 1.6 mV; The difference in AHP duration between adult-control cells (1.3 f 1.1 s; n = 7) and completelyadapting, adult-low BAPTA &Is (3.7 f 1.4 S; n = IO) was also significant. Medium and slow AHP amp& tudes and AHP duration were not sinnificantly altered in 1 week-low BAPTA neurons c&pared to 1 weekcontrol

adult-amtroi

cells (Fig. 3; Table

neurons

1).

3.8. Further comparison of firing Mtavior and Al-mill adult-low BAECA and l-week&f contml cells 3.7.2. Low BAPTA Adult-low BAPTA neurons tended to have larger and longer sAHPs than adult-control cells (Table 1, Figs. 2F and 3). When all the data were pooled for the and adult-low BAFTA cells (completely-adapting repetitively-firing cells), the differences between this group and the adult-control group did not reach statistical significance. If completely-adapting, adult-low BAPTA cells are considered alone, however, sAHP amplitude (7.5 f 4.6 mV, n = 10) was significantly

Al

The difterences we observed in firing and AHPs were not due to BAPTA- or age-induced differences in soike width or innut resistance of neurons in the differin spike ent groups. We controlled for diierences width by integrating (over time) the vobage area under all the spikes in a train. As previot& reported [9]. adult-control neurons shawed a linear increase in AHP data not shown) with induration (and amplitude: creased integrated area under the spikes during repeti-

A2

B2

Fig. 5. The phanneob of firins behavior and AHPs of adult-low BAITA cells was similar to that prcvim& duaibcd for 1 week- and adulI-coWrol rills 1% A: the p-noradremrgic a&mist isoprotercnol (LOOphi) reduced spike-frcqueacy&ptMion io adult-low BAITA alk. Firins W&Sevokedat RP by a 1.0 nA cwrcnt injection (1 S) for the control trace and a 0.75 QA in&&n for the iropmCcred mcord. Inset: the SAW was reduced by isoprotennol. AH& were evokedby 100 ms cunenl injections(1.25 nA for OXMIUIutd 1.0 nA for *opacrcnd to m&b the number of spikeselicited;9 spike.). B: the extremeadrpc~tion and large sAHF’of adult-lo* BAF’lX cellsmrc Ca-depcndeat.l’bis whdt-low WA cell WC&I not fire throughout the stimulusdunticn in control %oluticns(81). When a%rac&&r d+ MS trp*ad wilh Co*+ (2 ~Ilf@, adaptation and the sAHP were reduced and the ceil fired for the duration of the curren: injectioo(ez). Repetitivefirin# w8s CWkcd at m by I 1.0 aA carrent inject& (1 s) in both Bl and EL!.Inset: the sAHP was reducedwhen Cazt wa$rql&xdbyco*+.AHPSwe~ulktdbY20_2rm suprathresholdcurrent iw at 100 Hz. Gains for Al-B2 are as noted by the scak ban ia the center of the fi8uirurr. Gains for invts in Al and Bl are as no:d by the scalebaa in B! nut.

N.M. .Lmmam, RC. Fo&rin.g/&w~tal

tive activation (Pearson correlation coefficient = 0.815; Fii 4& see also [SD. Repetitively-firing neurons from l-week-old animals showed no relationship between AHP duration and spike area (correlation coefticient = 0.009; Fig. 4D). Completely-adapting l-week-old

cells showed a very slight negative correlation between AHP duration and spike area (correlation coefficient = -0.108, Fig. 4D, see also [SD. In the same cells, small spike areas could produce equivalent or longer AHPs than larger spike areas. High BAPTA impalements in the adult resulted in a

loss in the SAHP, there was almost no increase in AHP duratkm with increased spike area (correlation co&ciettt = 0.198, Fig. 4C). The relationship between integrated area and AHP duration in 1 week-high BAITA cells (correlation coefficient = 0.565; Fig. 4B) was different from that in the completely-adapting cells, but similar to that in the adult-control. Adult-low BAPTA cells, which did not have increased spike widths compared to adult-control cells, had AHP duration: area relationships (correlation coef6cient = -0.115) similar to that of the completeiyadapting, 1 week-control ceils @ii. 4F). The AHP duration: spike area relationship in 1 week-low BAP’I’A

cells also resembled that of the completely-adapting cells in the 1 week-control group (Fig. 4E). Prevkuuly, we have also shown that the differences between adult and 1 week-control neurons in AHP amphtude and duration were not due to differences in input resistance between the two groups [9]. When cells of similar input resistances were compared, completely-adapting immatur- neurons consistently had larger and longer sAI-IPs. Also, the age-related diierencc in the relationships between AHP amplitude or duration and integrated spike area was unchanged try normalization of the data by input resistance. Consistent with the previous study, in the present data, normalization by input resistance did not change the patterns for the AHP duration: spike area relationship in any experimental

3.8.1. I%amtacdogy The presented

group (data not shown).

of AHf% and jiring behavior data clearly suggest that tnodiications of AHPs and firing behavior were dependent upon age and chelator concentration. We then asked whether the mimicry of the immature, completelyadapting behavior by adult-low BAPTA neurons was due to the induction of novel mechanisms regulating the AHPs and firing, or to a greater relative expression of the same amductances determining these properties in control neurons. To differentiate between these altematives, we examined the effects of phatmacological agents known to affect the sAHP and spikefrequency adaptation in adult and immature neurons 191.As in control adult and immature nciuons. isopro-

Brain Resemrh M(1995) 192403

terenoi (100 PM; g-adrenergic agonist) decreased both the sAHP and spike-frequency adaptation in adult-low BAPTA cells (3/4 cells; Fig. 5A). These effects were

reversible (data not shown). Siiilarly, adaptation and the sAHP were reduced in solutions containing zero Ca2+/2 n&I Co2+ (5/5 cells; Fig. 5B). The similarity of responses to isoproterenol and Co2+ in adult-control, adult-low BAPTA, and 1 week-control cells suggests that no novel conductances were activated by the low BAPTA electrodes. Rather, adult neurons impaled

with low BAPTA electrodes mimicked immature completely-adapting cells in that both groups showed relatively greater expression of the same G-dependent currents, when compared to adult neurons impaled with KMeSO, electrodes.

4. BIscussIau We previously reported that a majority of control neurons from l-week-old animals exhibited extreme spike-frequency adaptation; neurons only fired for the first 100-250 ms of a 1 s current injection 191.The extreme spike-frequency adaptation in immature neurons was due to a greater relative expression of a Ca-depcncknt K+ current that was sensitive to modelation hy NE [9]. In the present study, we conilrmtd and extended these observations to include a more extensive comparison of layer III and V cells. We found a correlation between the proportion of completely-adaptating cells and cell layer. Previous studies have shown that neocortical development (in terms of cell biihdate and subsequent maturation) follows an inside-out gradient (e.g. [25D. and ceils located in the deep layers of cortex hecome motphologically mature before those in superficial layers (e.g. [5,2m. Dur finding that superiIcial cells more frequently exhiiit an immature firing pattern (complete adaptation) is consistent with such an inside-out developmental pattern. Several possible mechanisms could account for these age-reiated differences in C&dependent AHPs and Sring behavior (see Introduction). In the present study, we investigated the hypothesis that differences in Ca2+ regulation underlie the developmental changes in AHPs and fii behavior. We examined AHPs and firing in immature (l-week-old) and adult neurons with sharp micmelectmdes chelator, 2 mM

under three recording

conditions:

no

BAPTA, or 1tKHOOn&I BAITA. Our principal Endings were that in regard to AHPs and

firing behavior (1) adult-low BAPTA neurons mimicked 1 week-control cells, (2) 1 week-high BAPTA neurons were similar to ad&control cells, (3) a greater percentage of 1 week-low BAPTA neurons than 1 week-control cells showed complete adaptation, and

NM Lnrtmzm, RC. Fcwhrmg/LkwlopmmtaI (4) adult neurons

impaled with high BAPTA electrodes fired in a burst-spiking mode. We discuss these results in greater detail below. 4.1. High MA Many studies have utilized the chelating properties of BAPTA or EGTA to reduce Ca-dependent K’ conductances in adult neurons (e.g. [3,7,20,211X Reduction or loss of AHPs under these conditions is considered support for the hypothesis that the mechanisms underlying the AHPs are C&dependent. Consistent with the previously described effects of high concentrations of BAPTA on AHPs, we found that sAHPs were reduced in adult neurons impaled with high BAPTA electrodes, compared to control cells. Frequently, however, adult-high BAPTA cells fired in a phasic, burstspiking pattern rather than the tonic repetitive firing pattern seen in control neurons. This transition to burst-firing behavior has previously been described in guinea pig neocortex and has been attributed to reducinactivation of Ca*’ currents tions in C&dependent and C&dependent K+ conductances 131. High concentrations of BAPTA in l-week-old cells reduced sAHPs and spike-frequency adaptation. Thrse cells fired repetitively throughout long current injections (consistent with a reduction of Ca-dependent K+ conductances which contribute to spike-frequency adaptation; e.g. [91). No completely-adapting cells were observed in the 1 week-high EAPTA group. Thus, to a first approximation, altering intracelhllar CaZf buffering with high concentrations of BAPTA altered the properties of immature cortical neurons to mimic the adult conditions (although with high BAPTA impalements, AHPs were even smaller than adult controls and repetitive firing was sometimes compromised). 4.2. Low R4P?A We found that low concentrations of BAPTA (2 mM) in the recording electrode transformed the properties of neurons from adult rat neocortex to resemble those of immature neurons. Strong adaptation was seen in adult-low BAPTA cells. The mean amplitudes and durations of sAHPs, and degree of spike-frec;lency adaptation in adult-low BAPTA cells, were ali greater than in adult-control neurons and similar to those of 1 week-control cells. In general, these results reproduce in rat cortical neurons the anomalous results of low BAPTA reported by Schwindt et al. [21] in cat Betz cells. We extend those results by showing that low BAPTA electrodes allow adult cells to mimic the properties of immature nelnons. The effects of low BAPTA in immature neurons were more subtle. Without chelator in the electrodes,

Bratn Research84 f1995) 192-203

60% of neurons at 1 week of age shmved the completely-adapting pattern; the remaining celIs 6red repetitively. As a group, 1 week-low BAPTA cells were more likely to be cotnpIeteIy&apting (88% of cells) but had similar sAHP amplitudes and durations and percent adaptation to 1 weekcontrol neurons. Taken together, the low BAPTA data from adult and immature cells suggest that alterations in &+ buffering or regulation may underlie the differences in AHPs and firing behavior between normal adult and hnmature neurons. How the exogenous chelator affects the AHPs aad firing behavior is unknown, and interpretation of our results is complicated by our not knowing the actual intracellular concentrations of Caz+ or BAPTA. We assume that BAPTA levels were proportional to, but much lower than, pipette concentrations. Scbwindt and colleagues [213 suggested that the anomak~ behavior of low BAPTA could be expIained by a theorctical Ca” diffusion model proposed by sala and Hemandez-Cruz 191 (see also [15,16D. In that model, low concentrations of a fast, mobii buffer like BAPTA would be expected to decrease the peak [Cal,. but a small pool of BAPTA-bound Ca’+ would be established. Ca*’ couId then be released sIowIy &om this pool, thereby prolonging the CL?’ transient. Repetitive activation could allow summation of larger and more prolonged Ca” transients. Thus, low concentrations of BAITA could effectively facilitate the diffusikm of free Ca*+, and consequently allow Ca” to more effectiveIy reach the Ca-dependent K+ channels. Sii impakments with low EGTA elect&es produced f!ring behavior similar to low BAPTA el&mdes, the effect appears not to be depend on extremely rapid kinetics of chelation. Other possible mechanisms relate to steady-state Ca*+ levels. If 2 and 200 mM BAPTA buffered Ca’+ to different levels, cellular homeostasis might be biased toward processes with greater or lesser Ca*+ sensitivity. For example, &dependent inactivation of Ca*’ currents might be reduced by low BAPTA a~ce~trations, but (L-dependent K+ channels with high Caz+ affinity might still be activated. The complicated interactions of the various aspects of cellular Ca” buffering (binding proteins, internal stores, mitochondria! exchanges, Ca*+-ATF’ases, etc.) uptake, Na+-Ca*+ precludes definitive attribution of a mechanism to the low BAITA effect.

[

4.3. other celLr Completely-adapting immature neoaxkal ceils ~OIpaled with electrodes without chelator, and adult and immature cells impaled with low BAFTA ekchmles, have AHI’s and firing behavior similar to that of hip

lo2

h?Y Lomtzaii

RC F&ring/Dtwlapnmtd

pocampalCAI pyramid.alneurons (e.g. [llD. Intrinsic differencesin Caz+ metabolism,in addition to diierences in ionic conductances,may thus underlieat least part of the differencesin firingbehaviorbetween CA1 hippocampaland regular-spiking pyramidalcells of the neocortex[21]. Vagal motoneuronsalso demonstratea completely-adaptingfiring pattern and large, prolonged sAHP [18].In those cells, the sAHP is produced by Cadependent K+ conductanceswhich are gated by Ca2+ released from internalCa*+ stores [18].

Brain Research 84 (J99SJ 192-203

We gratefullyacknowledge the excellent technical assistance of K. Coffer. We are grateful to Drs. C.J. Wilson and W. Armstrong for valuable commentson an earlier version of this matmscript.This work was supported by NationalInstituteof NeurologicalDisorders and Stroke Grant NS-27188 (to R.C.F.], and an National Institute of Mental Health predoctoral fellowship (to N.M.L).

4.4. clmclusions Reference8 Severalpossible mechanismsfor explainingthe agerelated diierences in AHPs and firing patterns seem unlikelyin light of data obtained in this and previous studies. Data from both this paper and a previousone [9] suggest that the developmentaldifferencesare not cawed by differences in spike width or input resistance. The developmental presence of novel Ca-dependent K+ conductames at younger ages is unlikely giventhe pharmacologicalsimilaritiesof the AI-IF% and Sting in immatureand adult cells. The abilityof adult cells to mimicthe behaviorof the completely-adapting immaturecells when impaled with low BAlTA electrodes suggeststhat the relativedensity of Cadependant K+ channelsis unlikelyto be the major determinant of the agedependent diierenccs. The remaininghypotheses,suggestingdifferencesin Ca2’ in&u or a host of mechanismscollectivelyreferred to here as Ca2’ buffering,appear more likely. Preliminarydata suggest that the absolute density and amplitudeof high-thresholdCa2+ currents in acutely lsolated neocorticalneurons is much lower at 1 week than in the adult neurons[lo] (see also [SD.While it is possible that low BAPTA (or an endogenous buffer) increasesCa2+ inlluxby reducingCadepcndent inactivation or. Ca2’ currents,the low density of Ca2’ currents makes this an unliiely mechanism for the increase in Cadependent K+ currents in completelyadaptingimmaturecells. We favor the hypothesisthat Ca2’ buffering is qualitativelydifferent in immature and adult neurons. Finally,the profound influenceof calciumchelation on AHPs and llrlngbehavior,and the sensitivityof the underlyingCa-dependentK+ currentsto neuromoduMom such as NE, suggestsan intriguingpostbility for determinationof developmental critical periods for synaptic plasticity (e.g. [26D. Most models for such plasticityemphasizea thresholdfor synapticmodiications involvingCa2+ in&x through either voltage- or NMDA-gated Ca2+ channels [Ul. The present data suggest thai agedependent alterationsin Ca2+ regulatory mechanismscould also influence the timing of such criticalperiods.

111Connm’s, B.W., Gutnkk, MJ. and Frince, D.A., Ekctrophysiological pmpmtks of nwcmtkal neurons in vitro, I. Neumphysid.. 48 (1982) 13ts1320. [21 Finkel, AS. and Redman, SJ., Optiil voltage clamping with sin& mkmelectmde. la Smith, T.G. Jr., Lear. H., Redman, SJ. and Gap. P.W. (Ed,.), Vdtage and Patch CrcMpins with Miitmdes, American wysioloricsl Society, MD, 1985,95-120 pp. [31 Frkdman, A. and Gutnkk. MJ., Intracellular c&turn and control of bunt pncntiott in neurons of yaws-piu nwconex in vitro, Eur. J. NewwcL. l(19S9) 374-381. I41 Hamill. O.P., Huguctmrd, J.R. and Prince, D.A., Patch clamp studier of voltye gatcd atrrents in identified neurons of rat cen?bral cortex, crrrb. C&a, 1(1991) 1-6. 151Juraska, J.M. sod Fii E., A w study of the early patttatal development of the * cmtex of the hooded rat, 1. Camp. Ned. IS3 (1979) 247-256. [61 Ktie~ateitt, AR, Supper, T. and Prince, D.A., Celhtlrr and syllaptkp&ido#yandcpikp(qctleskofdev&pi~ratnee co&al tteumtts in vitro. Deu. Bmfn Res., 34 (19S7I 161-171. I7l K&sic, II, Pttil, E md Wetmatt, R.. EGTA and motoneumnal tier- potential& J. #%y&/., 27s w7l3) 199-223. ISILonuon, NM. and Foebring, R.C. Relationship between rcpet~iiw Rritts and aftcrltypcrpdarkatiom in human neomrtical tteutuos, I. h%un#tysid, 67 (1992) 350-363. 191Lonttzcm, NM. and Poebring, R.C., The ontwny of nptiiive firin and its modulatiott by ttorcpittePltrim in rat neoawtkal neumm, Den &pin Ra., 73 (1993) 213-W. [lo] Lercazrm, NM md Fceluiag, R.C.. pamauidcvclopneat of bisb*uuubddcakifrm currents ia acutely isolated rat scttsorimotor eortial ~umtts, SOS.Newwci. Ahsr., 19 (1993) 1127. [III Madison. D.V. and Nil, R.A.., Control of the teptitiw disdw#? of rat CA1 pymmidal tteutunea in vitro, 1. Fft@d. 354 w84) 319-331. [I21 McCormkk DA., Connors, B.W., Lixhthall, J.W. and Prince, DA., Cutt~uative ckcttwltysiohxv of pyramidal and ~pannely Ipioy stcllaIe ttcurons of the twocortex, J. Nrumphysid, 54 wS5) 782-1106. 031 MU%mkk, DA and Ptittcc. DA., Pat-natal tkvclopment of ekctm-phwkb&l propertics of ra1 cerebral pyramidal neutunes, 1. Bwtd.. 393 (1987) 743-762. 1141 Miller. M..~Matuntion of rat visual cortex. 1. A quantitative atudy of Gol#i-imprrgttated pynmidal ~~urotts, I. Newucytd, 10 (1981) 859-878. I151 N&X, E. lad Auattstine, GJ., Cakium #rad&ientsand buffers in btwitx chtwuft% cells, 1. &vid, 450 (1992) 273-301. 1161Navyelry. MC. and PittIer. MJ., Time umrses of calcium and cakittm-bwnd buffers folkwing cakium influx in a model cell, Bioprkys.J., 64 (1993) n-91.

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,

logxal expenments invitro?. Nd Len,

Fledman.