- -----.---.-x-I___ __..-- - -_-_ _ x.- _. .-. ---.. ._” ---“I .---

Both long- and short-term f’orms of’ synaptic pfaslicity are important cfluractcristics at’ cortical f~f~ysi~9f(9~y. l’hc~c phe~~omcn~t have been cx t cns ivc f y studied in IIIC hif>- p~9c~~l~~f~~lf ~ (~r i~~~ti(9n with respect to the cellular basis li9r memory [5, f8,23,42]. This research fras ~ocuscc i on estab- Iis fGng the mechanisms responsible for these phenomena. Hc9wcver . the apparent mcchnnisms va ry according to tfmc tyfjc of’ plasticity under investigation.

‘This paper deals with paired-pulse facilitation (PPfX a ft9rm of short-term synaptic pfi1sticity charuclcrized by iul enhanced neuronal exc itability atlcr a conditioning stimu- lus [4,9,19.2 f ,25,48]. Tflc mechanisms undcrfying PPF are thought to include an increase in the r c f cusc oL‘ neurolrans- mitter mediated by res idual free calc ium in OK tcrminalh activated by the c 0 ii d i 1 i 0 n i n g s lim u fus [3,12,19,2!5,33,48,49]. Direct ev idence fi9r cnfianced trans-

Rc c c lll Gndings suggc”s l that f~(9~lsyl~~f~lic ~ l~cc f l~~~i~l99~ may also f3fay an iiiiport~~~t role in PI-‘. ~ llldic~ wflicfs hate investigated the infsut-oulf3ul (f -0) rcf~~ti~9ll~fl~f~s in CA 1 indicate that a postsynaptic comf?oncnt contributes lo facilitation [22] und pf~~r~t~acofogicaf studies in the dentalc gyms suggest that NMDA receptors (911 the ~9oslsynaptis nicnihr;inc alsc9 contribute 1~9 ai enhanced test response [Hi].

G iven tflc c v idcncc fi9r a poslsynaptic ~~9rn~~~i~~~91 in I’PF, it s c ’c ’ms r txsC9nab)c that the f7 rcscncc ot’ an action

f7olcnli;~l in tfic postsynaptic neuron dlering lhc condition- ing response could influence the s cns itrvily ot’ lhis neuron to subsequent synaptic input. Tflis hypothesis is particu- fiirfy r c levailt in ligflt ot’ r c c cn l c v idcncc tier back-propik galion of somatic spikes into lhc dcndritic arbor of’ cotliCil1 ccf fs [3.l3.14.24,3S.39.4 I .46,.97]. Bccausc the ac~i011 p9-

tentid invxfcs the jcndritic rtrhor aficr the c c l1 has genur- alcd an oulf>ul spike. dcndritic spikes may bc more impor-

[ant various Finns of synaptic plasticity than in the pri- mary integration of synaptic inputs.

111 this study we used externally applied electric fields to investigate the contribution of action potentials to PPF in the dentate gyros. Field effects associated with this ~~~;~nipulation are known to alter the neuronal transmem- brane potential [7.8,10,32.34,45]. The fields were used to modify the number of cells discharging in response to a conditioning stimulus without influencing synaptic drive. If the presence of spikes during the conditioning response influences postsynaptic excitability at the time of the test response, this will be reflected as a change in PPF. On the other hand, a failure to influence the test response would suggest that spiking does not contribute to facilitation.

2. Materials and methods

Following a brief exposure to halothane anesthetic, male Wistar rats (150-2 g) were decapitated and the brain was removed. The hippocampus was dissected from the surrounding tissue using blunt dissection on a cold work surface (approx. 4°C). The isolated hippocampus was then placed on the chilled cutting surface of a tissue chopper and six to eight transverse slices were cut from the anterior-mid pole at 400 pm intervals. The slices were transferred by brush to the nylon net of a standard intcr- face chamber. The recording welt was perfused at 2-3 ml/min with oxygenated W!W O,, SVC CO?) artificial cerebral spinal tluid (ACSF) maintained at 34 it: 02°C. Slices were allowed I h to rccovcr following dissection.

The ACSF was it modified Ringer‘s solution cmkstiug of (in mM): 130 NaCI. 3.5 KCI, 1.25 NaH,PO,. I.S CiACl~, I .S MgSO,, 24 NaHCO,, iAnd IO II-glUCOSC. Before adding C&I2 the otherwise complete ACSF was bubbled with gas containing YS(;/r 8, and 5% CO, to avoid precipi- tation sf calcium.

Bipolar stimulation was applied to the surface of the slice via a pair of twisted 25 pm Teflon-coated platinum wires. The electrode was positioned with the aid of a dissecting microscope and a manual micromanipulator. A programmable timer and a ground isolated constant volt- age device were used to generate square stimuii with a duration of 0. I ms and an amplitude of I-30 V. Extracel- hltlr nlicrOpipettus here filled with 4 M NaCl and broken back to !i tip resistance of l-6 MJI. The electrodes were connected to a high impedance amplifier and referenced to a silver chlorided wire in the recording well. Signals were filtered (DC to 3 kHz) and then digitized by computer at a sample rate of IQ kHz and a resolution of 12 bits f(rr storage and subsequent analysis.

The recording electrode was placed in the upper blade

of the stratum granu depth was adjusted to ma tion spike amplitude (Fig. the perforant path was achieved b electrode in the outer two-thirds of t The stimulus intensity was adjusted un was half-maximal. Pairs of stimul s with an interstimulus interva interval was chosen based on results of a pi showing that an ISI of 50 ms produced maximal is consistent with the ISI used by o ing PPF [ 12.2 1,361. Slices that did tion spike, or slices that prod1 spikes in response IO a single stimulus, were rc.iecte

im experimental too to alter the somatic nmembmne poten-

STIMCLATL

4 SPIKE AMPLiTUDE

5 mV

WitI poGtioncd in the outer two-thirds of the mdCculitr layer. B: wavc- form imdysl!+. Sample p)pulittion spike recorrled from stratum granulo- sum. Spike amplitude was measured tram the peak posltlvlty (11 the EPSP to lhc peak ncgrrtivity of the spike. Presynuptic drive \vils dcter- mmed hy measuring the slope of the EPSP (dotted line).

c.

0 0 2 3 6 s 10

CONDITIOtilTG SPIKE RMPLITL’DE WI\‘)

80

40

6-l SPIKE

rA11’1 ITI ‘111

and f“eld intensity rcmaincd omskltrt thr~~u~t~~~ut the expor- imental protocol. The first condition was a control. In this trial, paired stimuli were delivered without applied fields to establish the amount of facilitation under control condi- tions. For the second and third trials. tlelds were applied during the conditioning response (Fig. 4A). A hyperpolar- izing field was applied during the second trial and a depolarizing field was applied during the third trial. In each ca.se, fields were initiated 10 ms before stimulation and remained constant for 30 ms. This ensured that the field was applied for the entire conditioning response but had terminated a full 30 ms before the test stimulus was delivered. At no time was a field applied during the test response. The test responses of trials 2 and 3 were com- pared to the test response of the control trial in order to assess any changes in PPF associated with alterations in conditioning spike amplitude (Fig. 4B).

The possibility exists that the applied field may have directly contributed to a change in the test response. In order to assess this possibility. two trials were performed in which fields were applied in the same manner as previous trials, but the conditioning stimulus was withheld. Since no conditioning response was evoked, any lasting effect of the field could be observed. At the end of the experimental protocol the response to a single stimulation was recorded in the absence of an applied field. This response was compared to the control conditioning spike in order to identify any changes in slice excitability that occurred over the course of data collection. If significant changes did occur, the data wcrc rejected,

Fig, 3. S-0 rclutmMip ia the t’ti\clil druttitit. A: ~l,rltph dl~a!td~~~ IIN R!lUtilXt4li~ bCtWCCl1 spike illll~lihld~ itlId EPSP hIOpt? ill incrming sJimuh8s intensities, In cuch slict?, muximill PS amplitude and uhsociatcd MPSP slope Wcrc dcfincd as lW%, All uthcr spike iqMudt2s utld

EPSP slopes wcrc nomalizcd with rcrspct to thcsc t~Mtd voluc~. ‘l’l~c grlrph Jrnmnslratcs that an incrcasc in spike mplitudc, resulting from incrcusinp stinrulus intunsity, is mmicwiatcd with P proportIona incrcusr in tEP!SP slope. The slope of this curve is approx. I. This graph i\l$o illustrates the method ysed to dctcrminc whether the 1-O rclutionship c~ln account for the increase in EPSP slqx obsorvcd during pr\ired-pulse stinwlution. Spike iunplitude of tbu conditioning response was SO% of maxitnul in itll paired-pulse trials (lower horizontcrl line). The itveriipc facilitation ub.scrvcd was 165%. This value for facilitrrtion Wilh uxd to &tcminc the matching location of the test spike amplitude on the 1-O curve (upper horizontal lint). Based on this value the matching EPSP 4opc WPS dctcrmincd (right vertical line). The difference bcrwccn the two vcrtio;ll lines ~ot~sponda to the f;lciliti\tiutl of the EPSP slq prcdictcd by the I-0 curve ( 171%). ‘MN point to the left of the dumb is the i\ctual PPF ck~~aci during paired-pulse tricrls. This clcwly illustr:~tcs the di+ cmpan~y Mwtin Ihe I-O and PPF data. t3: histogram funher illustrating the di.scnpmy between PPF kiuls and I-O data, The two bars on the left indicW the fxilitation of spike amplitude during paired-pulse stinrulalion und the matching spike amplitude dcscribcd in A. The bars on &,c right indicate tht? corresponding EPSP slopes. The diffcrcncc ktwccn the nh.Wncd fticilitation of tEP!SP slope ( 108 f 2%) imd that predicted by the I-O rAtionship ( I71 f l9%) is significant ( l P c 0.05).

A second set of animals was input-output (I-0) r~t~ltionshi~ of The same recording and stirnlil~~tin the PPF experiments. In order to csta ship the stimulus intensity was systematically increase from a level that did not produce a spike, to a level producing a maximal response. Groups of four were collected and averaged at each increment amplitude and associated fEPSP The objective for collecting these the change in fEPSP slop in spike amplitude when t increase in synaptic drive. T results from cad: normalized with respect (PS,,,,, ) and associated t amplitude was expressed

t

PPF Rcsponsr ---- ---------

60 so

fEPSP SLOPE (%)

Prcdickd

PPF I-O PPF I-O SPIKE fEPSP

AMPLITUDE SLOPE

CONTROL

The I-0 relationship of the dentate gyrus was investi- gated in order to determine whether the increase in presy- naptic drive observed on the test response during PP:’ was adequate to completely account for facilitation of the PS (Fig. 3). The I-0 curve was approximately h;.ar and exhibited a nearly equal incrcnsc in tEPSP slope with increasing PS amplitude. Based on the I-0 curves obtained from individual slices, an increase in the amplitude of a half-maximal PS of 167% was associated with an average increase in tEPSP slope of I7 I k 19%. This ~vits signifi- cantty greater than the facilitation of tEPSP slopes pro- duced by paired-pulse stimulation ( 108% ).

The contribution of action potential dischilrgc to post- synaptic mechanisms of PPF was investigated by using extcmnily applied electric fields to control the Ievcl of granule cell excitability during the conditioning response. The application of a hyperpolarizing field completely sup- pressed the PS in most cases and greatly reduced spike amplitude in the remaining slices. The avcragc spike am- plitudc of these suppressed slices was 18 f IX4 of the control conditioning response (Fig. 4A). Thcrc was no significant change in fEPSP slope. Rcvcrsing polarity 01 thC fiOld CUlNXt il Iilrgcr ncuronal ~N~pllliltiW~ to fire it1 rcsponsc to ~~fO~illlt pith StilllUlUtioil. ‘I’k! iiVCl7l$C in- cre’tsc in spike im~plitudc NU 136 f 1 I%. The tEPSP did show ii sligla! it~s;rst: in slope ( IUS & 2’~; 1 during applicrt- tion of dcp~3lurizing fir;ld!;.

Manipulating the number of cells firing action potcn- tick in response to the conditioning stimulus had no effect on the test response (f;lcilitation was asscssod with respect to the conditioning response of control trials) (Fig. 5). bpdurizing Gelds applied during the conditioning re- s~mse were associated with test response facilitation of 167 f I6%. The test facilitation associated with hyFrpo- hizing fields WZAS I60 f lS%. These viilues were not signiticantly different fmm the facilitation produced by puked-pulse stimulation under control conditions. The ~1.. cilitntian Of EPSP slope Was 107 f 2%” for trials with it hy~~~l~~~in~ tleld and 105) 4 2% for trials with (1 dcpo- h’ii?inp fidd. &spite large changes in spike ;brllplitu& during the conditioning response, tEPSP ~10~ and spike amplitude of the test response were equivalent to control Vducs (Fig. 5). This suggests that spiking activity does not intluence PPF.

\vhethcr the test rib tric fields applied durin g the ~~~n~it~~~f~in~ pwio ICl il

COSDII’IONIS’C; TEST RESPONSE RtSPohsi-.

t r; f

% c,

.-

The contribution of a postsynaptic me&anism in PPF has also been implicated by the findings of several other researchers, An analysis of the relationship between l-0 profiles and PPF in CA1 by Low et al. [221 reviled discrepancies similar to those observed in the present study. A current-source-density (CSD) analysis in the den- tate gyrus by Joy and Albertson [ 161 found that the den- dritic sink was not facilitated during the IEPSP. These findings are consistent with the present study and in each case the authors concluded that PPF involves both pre- and postsynaptic mechanisms. One important postsynaptic component may involve activation of NMDA receptors. Facilitation of PS amplitude is reduced in the presence ot NMDA blockers without intluencing the conditioning re- sponse [16). In addition the CSD analysis indicates an enhanced current-sink immediately following the test PS. Although this CSD finding is consistent with the NMDA receptor-coupled channel hypothesis it is also possible that inhibitory currents contributed to this enhancement 13 I].

There is alsa evidence that granule cells may remain depolarized after synaptic input for sufficient time to allow synaptic excitation associated with the test stimulus to superimpose on the sustained conditionwg depolarization (see Fig. la of [4]J. This would increase the number of active neurons participating in the test spike through sim- ple temporal summation. However, most intracellular stud- its report the presence of IPSPs at short latcncics [ 1 I ,27,2Hj, thus this possibility remains controversial.

In order to further invcstigutc possible postsynaptic mechanisms involved in PPF. the offcot of spiking \W synuptic efficacy was cxamincd. Somatic action polentiuls are major events in the postsynaptic cell. It is reasonable that an event at’ this magnitude could alter the pWsynaptic environment and affect the response of a neuron to subse- quent stimulation. The possibility uf spike-induced changes is particularly relevant in light of evidence for dendritic spikes in neooortcx [4 I ], hippacampus [3~13,2435,39.46,47] UI~ dentate gyrus f I$]. These den- dritio spikes may be important in both long- and short-term ~CWI~~S of synaptic plasticity. As a somatic action potential invades the dendritic arbor it, in effect, broadcasts the fact that the cell has initiated an output spike, This may prime the dendrite for facilitation by directly uiterinp rL\wptw- channel complexes or other membrane properties.

To establish the contribution of spikes to PPF we used an experimental paradigm which was c;tp&le of in&pn- dently influencing cell discharge and synaptic drive. Ap- PIid fields were used as a tool to control the PS amplitude

depolarizing field will increase rons and increase the PS amptitudc hypespolarizing field will decrease the if sufficiently intense, complete

The presence of a conditioning spike res~~~ti~~~ from t first of the paired stimuli did not i~~luet~ce the test re- sponse. Both the responses were equi delrilwlslrutes that dendritic spikes, ~~tterc manner necessary that manipulation ( results; of this stu cxpccted that inh tioning spike was increased by a dep~~~itri~i inhibition would be small when there was no c~~nditio~in~ spike during the hyperpolarizing field. However, the find- ing that test response PS amplitudes were equivalent for a11 trials suggests that inhibition was not ill~luenced magnitude of the conditioning spike. In additioti, recurrent inhibition is maximal for ISIS less than 25 ms following a conditioning response [I X37,40]. The for this experiment is long enough that recurrent it~~ii~~ili~~1~ would be minimal during the test response.

The finding that postsynaptic action potentials bavc INI &Tcc~ OII PPF has impt ml implications for siblc rold” 01’ ~te~~dritic ~Pi~c~ tlcllrl)~~i~t ~~~~~~~~~it~~~ %,I be vicwcd in the context 01‘ possible functions 01’ voltage gatcd dcndritic conductancc~. The result5 of ccl I attached patch recordings in CA1 apical dendrites indicate thut voltage gated channels WC activated during EPSP and AP propag:ttion [24]. A fundamental role of these channels may be to actively boost the electrotonic propagation of postsynaptic potentials necessary for dcndritic integration of distal inputs. A back-propagating dendritic spike will depolarize the dendritic arbor substantially above the volt- age of an underlying EPSP and should further activate voltage sated channels. The ultimate infhwxe at’ this additional activation on subsequent synaptic inputs wiil depend on channel kinetics and a variety of membrane properties [X3]. Some channel types may quickly return to their resting state while others may remain active long enough to contribute an additional boost to postsynaptic potentials arriving after the neuron has fired an initial output spike.

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