VC

5 0 -5 -10 -15 -20 -250

4

8

12

16

20

-10 0 10 20 30 40 50

0

4

8

12

16

20

-5 0 5 10 15 20 25 30 350481216202428

-40

-30

-20

-10

0

0

10

20

30

40

0

10

20

30

40

50

RC VCPP VC

RC pas.V

C

PP pas.V

C

RC VCPP VC

RC pas.V

C

PP pas.V

C

RC VC

PP VCRC pa

s.VC

PP pas.V

C

VC RC VC PP Pas.VC RC Pas.VC PP

Passive VC (Ca2+, Na+, K+channels blocked)A

C

D

E

B

0 10 20 30 40 50 60 70020406080100

0 10 20 30 40 50020406080100

0 -10 -20 -30 -40 -50

020406080100

No

of e

vent

s

10

20

30

40

RC PP020406080100

REC

A B

C

D-10 0 10 20 30 40 50

051015202530

RC PP

-20 0 20 40 60 800

4

8

12

16

20

0.0 0.2 0.4 0.6 0.8 1.00

20

40

60

80

100

10 20 30 40 500

2040

60

80100

0 20 40 60 80

020406080100

-0.2 0.0 0.2 0.4 0.6 0.8 1.0024681012

Rise (ms)

AMPA uEPSP from RC and PP

AMPA uEPSC from RC and PPLinear fit of RC uEPSP/SCLinear fit of PP uEPSP/SC

PP uEPSP/SCRC uEPSP/SC

0 20 40 60 80 100 120-30

-25

-20

-15

-10

-5

0

0 4 8 12 16 20 24 280

-5

-10

-15

-20

-25

-30

0 4 8 12 16 20 24 280

20

40

60

80

0 5 10 15 20 25 30

10

20

30

40

50

4 8 12 16 20 24 4 8 12 16 20 24 280

20406080

100120140

5 10 15 20 25 3020

40

60

80

100

0 40 80 120 1600.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0a b c d

e f g h

0

20

40

60

80

100

120

0

20

40

60

80

RC PP

0.1 mV

B

C

D

S1 (RCstim.)

S2 (PPstim.)

REC

A

25 ms

0 40 80 120 1600

3

6

9

12

15

18

21

0.0 0.1 0.2 0.3 0.4 0.50

3

6

9

12

15

18

0 20 40 60 800

3

6

9

12

15

18

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.70

20

40

60

80

100

20 40 60 800

20

40

60

80

100

0 40 80 120 1600

20

40

60

80

100

A

5 pA10 ms

5 pA5 ms

- 64 mV - 64 mV - 65 mV - 65 mV

B

-20 0 20 40 60 80 1000

5

10

15

20

25

-20 0 20 40 600

5

10

15

20

25

0 -2 -4 -6 -80

5

10

15

20

25

VC

-2 -4 -6 -8 -10 -12

020406080100

0 20 40 60 800

20

40

60

80

100

0 20 40 60 80 1001201400

20

40

60

80

100

C

D

E

HH

W (

ms)

Dec

ay (

ms)

Dec

ay (m

s)

NMDA uEPSP from RC and PP

NMDA uEPSC from RC and PP

0 40 80 120 160 2000.05

0.15

0.25

0.35

0.45

0 100 200 300-12

-10

-8

-6

-4

-2

0

5 15 25 35 450.05

0.15

0.25

0.35

0.45

5 15 25 35 4520

60

100

140

0 10 20 30 40 500

-2

-4

-6

-8

-10

-12

0 10 20 30 40 50

0

50

100

150

200

250

0 10 20 30 40 500

20

40

60

80

100

120

0 10 20 30 40 50

0

50

100

150

200

250

CA3 pyramidal neurons (CA3pns) receive stratified inputs from three principal sources (Figure 1): in the stratum lucidum the mossy fiber pathway (MF), in the stratum lacunosum molecularis the perforant pathway (PP), and in the stratum oriens and radiatum the re-current collaterals (RC) inputs from other CA3pns located ipsilaterally and contralaterally.The aim of this study was to characterize AMPAR- and NMDAR-mediated somatic unitary responses evoked in CA3bpns after stimulation of RC and PP inputs. Synaptic responses were evoked with a minimal stimulation protocol in current clamp (CC), volt-age clamp (VC), and passive voltage clamp conditions (passive VC).

Preparation: in vitro slices from male Sprague-Dawley rats (19- 28 days old).

Electrophysiological techniques: Whole-cell current clamp and voltage clamp recordings from CA3pns were obtained with the aid of infrared DIC video microscopy.

EPSPs and EPSCs, were evoked by minimal stimulation using extracellular stimulating electrodes (concentric bipolar CBAPC100; FHC).

Drugs used: bicuculline, 10 µM and CGP35348, 500 µM were added to the perfusion bath to block GABAA/GABAB receptors, re-spectively. The AMPAR- and NMDAR-mediated unitary components were isolated with the addition of D-APV (50 µM) or CNQX (20 µM), respectively.Synaptic responses were obtained under three different conditions: 1) Current clamp; 2) Voltage clamp; and 3) Passive voltage clamp (pipette solution contained Cs+ and QX-314 to block K+ and Na+ currents; Ni2+ was added to the bath to block Ca2+ cur-rents).

Data analysis: using custom developed software that identifies single synaptic responses from failures and combined responses and computes the values of the different parameters used in the quantitative characterization of unitary synaptic responses such as peak value (PV), time to peak (TTP), and half-height width (HHW) (Figure 2). The expected number of unitary responses, given the observed failure rate, was calculated using a Poisson distribution. Based on this estimation, “putative unitary” responses with the lowest peak amplitude values were selected up to the number of responses expected from the Poisson distribution.

Statistical analysis: Statistical significance was asset using one- way ANOVA tests.

Introduction

Methods

A. Position of stimulation and recording electrodes. S1 and S2 are the sites to stimulate PP and RC, respectively. The record-ing electrode (REC) is in one CA3bpn. B. Unitary responses from RC (left traces) and PP (right traces). Average trace of RC (left) and PP (right) EPSP successes. PV (left), TTP (center) and HHW (right) histograms (C) and cumulative probability distribu-tions (D) for RC AMPA uEPSPs (blue), and for PP AMPA uEPSPs (gray). Histograms do not include failures. Insets: Plots showing the mean and standard deviation of the unitary values (circle) of different uEPSCs parameters in both recording conditions VC and passive VC.

AMPAR-mediated uEPSPs from RC and PP inputs

NMDAR-mediated uEPSPs from RC and PP inputs

AMPAR-mediated uEPSCs from RC and PP recorded under voltage clamp (VC) and passive VC conditions

A. Position of stimulation and recording electrodes. S1 and S2 are the sites to stimulate PP and RC, respectively. The recording electrode (REC) is in one CA3bpn B. Unitary responses from RC (left traces) and PP (right traces). Average trace of RC (left) and PP (right) EPSP successes. PV (left), TTP (center), and HHW (right) histograms (C) and cumulative probability distributions (D) for RC NMDA uEPSPs (blue), and for PP NMDA uEPSPs (gray). Histograms do not include failures. Inset: Plots showing the mean and standard deviation of the unitary values (circle) of different uEPSP parameters.

Unitary responses from RC (left traces) and PP (right traces) recorded in VC (A) and in passive VC (B), respectively. Average trace of RC (left) and PP (right) EPSCs successes recorded in VC and in passive VC, respectively. PV (left), TTP(center) and HHW (right) histograms (C) and cumulative probability distributions (D) for RC AMPA uEPSCs re-corded in VC(gray), and in passive VC (blue), and for PP AMPA uEPSCs recorded in VC(red) and in passive VC (green). Histograms do not include failures. E. Plots showing the mean and standard deviation of the unitary values (circle) of different uEPSCs parameters in both recording conditions VC and passive VC.

Unitary responses from RC (left traces) and PP (right traces) recorded in VC (A) and in passive VC (B), respectively. Average trace of RC (left) and PP (right) EPSCs successes recorded in VC and in passive VC, respectively. C. PV (left), TTP (center) and HHW (right) his-tograms (C) and cumulative probability distributions (D) for RC NMDAR uEPSCs recorded in VC (gray), and in passive VC (blue), and for PP NMDAR uEPSCs recorded in VC (red) and in passive VC (green). Histograms do not include failures. E. Plots showing the mean and standard deviation of the unitary values (circle) of different uEPSCs parameters in both recording conditions VC and passive VC.

NMDAR-mediated uEPSCs from RC and PP recorded under voltage clamp (VC) and passive voltage clamp (passive VC) conditions

Amplitude dependence and filtering of EPSP/EPSC kinetic

Scatter plot of the amplitude of AMPA (a, b, e, f) and NMDA (i, j, m, n) somatic unitary responses from RC and PP: 1)as a function of rise time, recorded in CC (a, i) or in VC condition (e, m), and 2) as a function of decay time, recorded in CC (b, j) or in VC condition (f, n). Relationship between the HHW and rise time of AMPA (c, d) and NMDA (k, l) somatic unitary re-sponses recorded in CC (c, k) or in VC condition (g, o). Relationship between the decay time and rise time of AMPA (d, h) and NMDA (l, p) somatic unitary responses recorded in CC (d, l) or in VC condition (h, p). The linear fit asses correlations be-tween measured parameters.

i j k l

m n o p

Fig.1. Schematic diagram of CA3 area showing the location of CA3 pyramidal neurons and of the synaptic inputs from MF, RC, and PP. Abbreviations: A, Alveus; SO, s.oriens; SP, s. pyramidale; SL, s. lucidum; SR, s. radiatum; SL-M, s. lacunosum-moleculare.

PP

RC

pn

RC

Cel

l num

ber

c23

c50

c98

c99

c116

10 20 30 400 20 40 60 8000.4 0.6 0.8 1.00 0.2

c18

c23

c50

c98

c99

c116

c18

c23

c50

c98

c99

c116

c18

E

Passive VC (Ca2+, Na+, K+ channels blocked)

Current Clamp

Voltage Clamp

Mea

n P

V (m

V)

Distance (µm)

Mea

n TT

P (m

s)

Distance (µm) Distance (µm)

Mea

n H

HW

(ms)

RC radiatumLinear Fit of Data

RC oriens

B C

REC

A

S1

S2

5 pA

AMPAR-mediated uEPSPs and uEPSCs from radiatum RC and oriens

Radiatum Oriens Radiatum Oriens

Oriens

S2

Radiatum

S1

D

E

MF

HHWPV

TTP PV

TTP

HHW Fig. 2. Synaptic parameters measured from EPSPs/EPSCs: PV; peak value, TTP; time to peak, HHW; half height width.

CC VC

A. Position of stimulation and recording electrodes. S1 and S2 are the sites to stimulate the radiatum RC (150- 300 µm from stratum pyramidale) and oriens RC, respectively. The recording electrode (REC) is in one CA3bpn. AMPA uEPSPs (B) and uEPSCs (C) from RC stimulated on the radiatum (left traces) and RC stimulated on oriens (right traces) and their corresponding average at the bottom. Scatters plots of the mean PV (left), mean TTP (center), and mean HHWs (right) of the somatic uEPSP (D) and uEPSC (E) versus the stimulation distance measured from the soma.

We used a minimal stimulation protocol and a Poisson estimate to characterize the AMPAR- and NMDAR-mediated uni-tary components for PP and RC inputs to CA3pns in three different experimental conditions: current clamp (CC), voltage clamp (VC), and passive voltage clamp (passive VC). In each of these conditions, we measured peak value (PV), time to peak (TTP), and half-height width (HHW).

1. The mean PV of AMPAR RC unitary response was significantly larger than that of AMPAR PP unitary responses in all con-ditions (CC, VC, and passive VC). However, the difference between PVs was less than the theoretical expected difference from passive models (see Soc. for Neurosci. poster 44.5) which show that PP AMPAR synaptic responses may overcome cable attenuation with increased conductance.

2. The difference in PVs between RC and PP was bigger in the passive VC condition than in the other two conditions. This would suggest that voltage dependent conductances are involved in the shaping of PP responses (Urban et al., 1998).

3. The mean PV of NMDAR RC unitary responses was significantly larger than that of NMDAR PP unitary responses in CC condition, but in the passive VC condition that difference was abolished indicating that also some voltage dependent conductances might be involved.

4. The kinetic parameters (TTP and HHW) of the unitary AMPAR responses followed the cable theory predictions: latency and duration were slower for the more distally located PP synapses.

5. There were not differences in the kinetic parameters of the NMDAR unitary responses between the two inputs in the CC and VC condition. However, in the passive VC condition, the TTP was longer for PP synapses, which could be explained by NMDA signals becoming faster in the passive VC, and consequently undergoing more cable filtering.

6. AMPAR- and NMDAR-mediated somatic unitary responses are subject to cable filtering, however that does not account for the PV variability of somatic unitary responses.

Summary and Conclusions

SUPPORTED BY: NIH GRANT AG025633

0.2 mV10 ms

5 pA10 ms

10 pA10 ms

44.4/T10Quantitative characterization of AMPAR- and NMDAR-mediated somatic unitary synaptic inputs to hippocampal CA3 pyramidal cells

T. Perez1, J.L. Baker2, M. Ferrante2, G.A. Ascoli2,3 and G. Barrionuevo1. 1. Dept. of Neurosci., Univ.of Pittsburgh, Pittsburgh, PA; 2. Krasnow Inst. for Advanced Study, George Mason, Univ., Fairfax, VA; 3. Mol. Neurosci. Dept., George Mason, Univ., Fairfax, VA.

-10

-8

-6

-4

-2

0

010203040506070

020406080100

-100 0 100 200 300-25

-20

-15

-10

-5

0

-100 0 100 200 3000

4

8

12

16

20

-100 0 100 200 30005101520253035

-100 0 100 200 300 4000.00.10.20.30.40.50.60.70.8

-100 0 100 200 300 4005

10

15

20

25

30

-100 0 100 200 300 40010

20

30

40

50

60

70

0.2 mV10 ms

5 pA10 ms

Perforant path

0.2 mV10 ms 10 ms

Mea

n P

V (p

A)

Mea

n TT

P (m

s)

Mea

n H

HW

(ms)

Distance (µm) Distance (µm) Distance (µm)

PV (mV) TTP (ms) HHW (ms)

No

of e

vent

s

No

of e

vent

s

PV (mV) TTP (ms) HHW (ms)

Mean PV (mV) Mean TTP (ms) Mean HHW (ms)

Cum

. Pro

babi

lity

Cum

. Pro

babi

lity

PV

(mV

)

TTP

(ms)

HH

W (m

s)

0.0.20.40.60.81.0

RC PP 0

Cum

. Pro

babi

lity

Cel

l num

ber

Cel

l num

ber

RC PP

Perforant path

No

of e

vent

s

No

of e

vent

s

No

of e

vent

s

0.

0.1

0.2

0.3

0.4

0.5

0.6

RC PP

PV

(mV

)

TTP

(ms)

HH

W (m

s)

RC PP RC PP

Cum

. Pro

babi

lity

Cum

. Pro

babi

lity

Cum

. Pro

babi

lity

Perforant path

S2 (PPstim.)

S1 (RCstim.)

RC PP RC PP

No

of e

vent

s

No

of e

vent

s

No

of e

vent

s

Cum

. Pro

babi

lity

Cum

. Pro

babi

lity

Cum

. Pro

babi

lity

PV

(pA

)

TTP

(ms)

HH

W (m

s)

RC PP

PV (pA) TTP (ms) HHW (ms)

PV (pA) TTP (ms) HHW (ms)

VC RC VC PP Pas.VC RC Pas.VC PP

RC PP RC PP

120

No

of e

vent

s

No

of e

vent

s

No

of e

vent

s

Cum

. Pro

babi

lity

Cum

. Pro

babi

lity

Cum

. Pro

babi

lity

PV

(pA

)

TTP

(ms)

HH

W (m

s)

PV (pA) TTP (ms) HHW (ms)

PV (pA) TTP (ms) HHW (ms)

- 63 mV - 62 mV

- 80 mV- 80 mV

RC PP

- 80 mV - 80 mV - 80 mV - 80 mV

PV

(mV

)

PV

(mV

)

HH

W (m

s)

Decay (ms) Rise (ms) Rise (ms)

PV

(pA

)

Rise (ms)

PV

(pA

)

Decay (ms)

HH

W (m

s)

Rise (ms) Rise (ms)

Dec

ay (m

s)

PV

(mV

) P

V (p

A)

Rise (ms)

Rise (ms)

Decay (ms)

Decay (ms)

PV

(mV

) P

V (p

A)

HH

W (

ms)

Rise (ms)

Rise (ms)

Dec

ay (

ms)

Rise (ms)

Rise (ms)

- 80 mV- 80 mV- 80 mV- 80 mV

Linear fit of RC uEPSP/SCLinear fit of PP uEPSP/SC

PP uEPSP/SCRC uEPSP/SC

*** *** ***

*** ***NS NS

*** ***

***

*** ***

*** *** ***

*** ***

*** ****** ***

Reference:

Urban NN, Henze DA, Barrionuevo G (1998) Amplification of perforant-path EPSPs in CA3 pyramidal cells by LVA calcium and sodium channels.J Neurophysiol 80:1558-1561.

PV (mV) TTP (ms) HHW (ms)

PV (mV) TTP (ms) HHW (ms) RC VCPP VC

RC pas.V

C

PP pas.V

C

RC VCPP VC

RC pas.V

C

PP pas.V

C

RC VCPP VC

RC pas.V

C

PP pas.V

C