Slower spontaneous excitatory postsynaptic currents in spiny versus aspiny hilar neurons

Neuron,

Vol. 8, 745-755,

April,

1992, Copyright

0 1992 by Cell Press

h CHurrents in Spiny versus Aspiny Hilar Neurons Charles T. Livsey and Stefano Vicini FIDIA-Georgetown Institute for the Neurosciences Georgetown University School of Medicine Washington, D.C. 20007

Summary In the hilar region of the rat hippocampus, large spontaneous excitatory postsynaptic currents (sEPSCs) mediated by non-NMDA glutamate receptors are present in both excitatory spiny mossy cells and inhibitory aspiny hilar interneurons, making these neurons ideal candidates for a comparative study using the tight seal whole cell recording technique. Although sEPSCs have similar amplitude distributions, the rise and decay times are significantly slower in spiny versus aspiny neurons. Similar kinetic differences are observed in synaptic currents evoked by mossy fiber stimulation. These results demonstrate a physiological difference between the excitatory drive to excitatory and inhibitory neurons in the hilus that certainly contributes to differences in synaptic strength and that may be applicable to other brain regions. Furthermore, since the development or modification of individual spines or groups of spines may affect synaptic strength, these results may be pivotal in establishing a role for spines in modulating synaptic activity. Introduction Neurons of the rat hippocampal hilus are strategically located between the dentate gyrus and region CA3, where they are thought to regulate hippocampal activity (Scharfman et al., 1990; Scharfman, 1991) and to be involved in the pathogenesis of epilepsy (Scharfman and Schwartzkroin, 1990; Sloviter, 1987). The complex circuitry of the hilus contains neurons covered with spines and thorny excrescences, spiny mossy cells (SMCs), and neurons totally devoid of spines, aspiny hilar interneurons (AHIs). Axon collaterals from en passant mossy fibers arising from dentate granule cells provide excitatory synaptic input to SMCs and some AHls as the main mossy fiber branches transversethe hilustoCA3pyramidal neuronsofthe hippocampus (Amaral, 1978; Ribak and Seress, 1983; Ribak et al., 1985). Additional excitatory inputs to neurons of both classes have recently been reported in adult rats, from the entorhinal cortex via the perforant path (Scharfman, 1991). AHls and SMCs in turn project to a variety of targets in the ipsilateral and contralateral fascia dentata (Blackstad, 1956; Zimmer, 1971; Swanson et al., 1978; Laurberg and Sorensen, 1981). It is well known that the mossy fibers provide a primary excitatory input to CA3 pyramidal cells, and neurotransmission at these synapses is thought to be mediated primarily through glutamate acting on non-N-methyl-o-aspartate (NMDA) receptor-gated

ion channels (Storm-Mathisen and Ottersen, 1989). Anatomically similar synapses occur on the thorny excrescences (complex spines) located on the proximal dendrites and cell bodies of SMCs (Amaral, 1978; Ribak et al., 1985), and large mossy fiber-like synaptic expansions have been observed in apposition to smooth cell bodies and dendrites in the hilus (Amaral, 1978; Ribakand Seress, 1983). However, the physiological and pharmacological properties of hilar synapses have not been examined as thoroughly as those in CA3. Stimulation and intracellular recording from pairs of hilar neurons have confirmed connections from granule cells to SMCs, granule cells to AHls, AHlstogranulecells,AHlstoSMCs,andAHlstoAHls (Scharfman et al., 1990). The output of granule cells was found to be excitatory, whereas that of AHls was inhibitory. The output of SMCs remains unclear, since these neurons, at least in some studies, display immunoreactivity for glutamate (Fischer et al., 1986; StormMathisen et al., 1983) and are thought to provide significant commisural input to the dentate, which is primarily excitatory (Deadwyler et al., 1974), but were found to make symmetric synapses (which generally indicates inhibitory action) with interneuron dendrites (Scharfman et al., 1990). Thus, given the current understanding of hilar circuitry, the excitatory synaptic connections to the AHls and SMCs in the hilar region seem quite similar. Interestingly, intense and continuous spontaneous excitatory synaptic activity was also reported in both neuronal types (Scharfman et al., 1990; Scharfman, 1991), similar to observations made in CA3 neurons (Brown et al., 1988). In the present paper, we investigate thecharacteristics of excitatory postsynaptic currents (EPSCs) in SMCs and AHls using the high resolution whole-cell patch-clamp technique (Hamill et al., 1981) from brain slices (Edwards et al., 1989). Spontaneous EPSCs (sEPSCs) occur with high frequency in both SMCs and AHls, thus providing large samples of comparable synaptic events from a wide range of membrane locations from spiny and aspiny neurons. We demonstrate that fast or slow kinetics of non-NMDA receptor-mediated sEPSCs correlate well with the absence or presence of postsynaptic spines, respectively. We provide evidence that these differences in kinetics do not arise simply from dendritic filtering of the synaptic currents, but are likely due to filtering by the dendritic spine and/or differences in non-NMDA glutamate receptor subtypes in the two neuronal populations.

Electrophysiological and Morphological Characterization of Hilar Neurons At least nine morphologically distinct neuronal types with a large variety of shapes and sizes are present in the selected region of the rat hippocampal hilus

NeLlrOll 746

50 ms 20 mV I200 pA

Figure

1. AHls

and

SMCs

Have

Distinct

Electrophysiological

and Morphological

Characteristics

In this representative AHI (A), a depolarizing current injected through the somatic recording electrode (resting potential, -62 mV) evokes a fast rising depolarization and repetitive AP firing, each followed by a large afterhyperpolarization. Depolarization of an SMC (B) (resting potential, -65 mV) by current injection evokes a slower rising depolarization followed by several APs, each being followed by a slight afterpolarization. (a) Confocal microscopy photograph of a Lucifer yellow-injected AHI and SMC demonstrating the absence of spines on the proximal dendrites and cell body on the AHI and their presence on the SMC. (b) Higher magnification micrographs demonstrating the smooth proximal dendrites of the AHI and the thorny excrescences on the large proximal dendrites of the SMC. Bars, 20 urn for (a) and 5 urn for (b).

between the dentate gyrus and the beginning of the CA3 pyramidal cell layer (zone 4, Amaral, 1978), but physiologically they can be lumped into two broad classes (Scharfman et al., 1990), those with action potentials (APs) followed by large (at least IO mV) afterhyperpolarizations (AHls, Figure IA) and those with APs followed by small afterpotentials (SMCs, Figure IB). Confocal microscopy inspection following Lucifer yellow injection of 14AHls and 15SMCs revealed AHls to be varied in shape and size of the dendritic tree and devoid of spines on the soma and dendrites (Figure IA, a and b), while SMCs had a fairly regular shape, each being multipolar with three or four primary den-

drites

covered

with

thorny

excrescences

and

distal

dendrites bearing simpler spines (Figure IB, a and b). The soma of many SMCs was also studded with complex spines (as reported by Amaral, 1978). The presence or absence of spines in 14 to 26-dayold rats correlated well with AP characteristics of SMCs and AHls, respectively. However, in younger rats spineswerefound in 2 neuronselectrophysiologitally classified as AHls, and it was more difficult to distinguish AP characteristics. In 13 of 13 SMCs and 11 of 11 AHls from animals older than 13 days, AP characteristics correlated well with morphology. Current-clamp recordings from II2 hilar neurons of this

Synaptic 747

Currents

in Hilar Neurons

a

b-

-

Figure 2. TTX-and Mn’+-InsensitivesEPSCsfrom AHIsand Were Abolished by a Non-NMDA Receptor Antagonist

SMCs

(A) (a) Series of SEPSIS in the presence of TTX and Mn’+ from an AHI. (b) Blockade of sEPSCs by CNQX (IO )rM). All sEPSCs were abolished within 30 s from the beginning of bath perfusion with CNQX. (c)An example of the fitting analysis of one sEPSC from this AHI. The decay time constant of the exponential curve was 2.0 ms. (B) (a) Slower sEPSCs from an SMC. (b) Blockade of sEPSCs by CNQX (IO PM). (c) Example of the fitting analysis of 1 sEPSC from this SMC. The decay time constant of the exponential curve was 9.8 ms.

age group revealed 60 neurons with characteristics consistent with the SMC definition and 52 neurons matching the AHI classification. The average resting potential for AHls was -49 + 7 mV, and the input resistance was 315 f 54 Ma; SMCs had a resting potential of -52 k 8 mV and an input resistance of 257 k 43 MS1 (mean + SD).

Glutamate-Mediated

sEPSCs

Whole-cell voltage-clamp recordings from hilar neurons revealed large (range 4-160 PA), high frequency (range 2-80 Hz) sEPSCs not abolished by the presence of 1 PM tetrodotoxin (TTX) and 2 mM MnCl2. These currents likely resulted from spontaneous vesicular release of neurotransmitter at somatic and dendritic synapses, in analogy to the release of acetylcholine at nicotinic synapses at the neuromuscular junction (Fatt and Katz, 1952). In both AHls and SMCs (Figure 2, a and b traces), currents recorded in the presence of TTX and MnC12 (V,, = -60 mV) were completely abolished by the nonNMDA receptor antagonist (Honore et al., 1988) 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 PM; I5 AHls and 20 SMCs), and in most cells, the decay phase of each sEPSC could be fitted by a single exponential function (Figure 2, c traces). Amplitudes and kinetics were not affected by picrotoxin (20 PM), a y-aminobutyric acid type A receptor antagonist (4AHIsand5SMCs),nor bytheNMDAreceptorantagonist (Davies et al., 1986) 3-[(+)-2-carboxypiperazin-4 yl]-propyl-lphosphonic acid (CPP, 10 PM; 10 AHls and 13 SMCs). In 1 AHI, large, slow sEPSCs remained

after CNQX but were abolished by CPP, and in 5 SMCs and 3 AHls, the decay phase of some sEPSCs was best fitted by the sum of two exponential functions (data not shown). Neuronsdisplaying sEPSCswith twocomponents at vh = -60 mV were excluded from further analysis. The most intriguingfindingwas that sEPSCs in AHls (n = 48) were remarkably fast in rise time (0.5 + 0.2 ms) and decaytimeconstant (3.6 + 1.5 ms), but sEPSCs in SMCs (n = 55) were slower, having a rise time of 1 .O + 0.3 ms and a decay time constant of 9.6 + 4.8 ms. The differences in average rise and decay kinetics between cell types were statistically significant (Student’s t test for grouped data, p < 0.01). The differences were similar for neurons classified according to morphology: AHls (n = II), rise time = 0.5 & 0.1, decay time constant = 3.7 + 1.0; SMCs (n = 13), rise time = 1.0 & 0.3, decay time constant = 8.7 i 3.0. Rise time and decay time constant frequency histograms were constructed for each SMC and AHI in order to analyze not only average sEPSC kinetics, but the entire range from fast to slow events. When the entire population of sEPSCs in each cell was accounted for, the differences in kinetics were still obvious (Figure 3A versus 3B, a and b graphs). In the SMC shown (Figure 3B, b graph), no decay time constant was faster than 4.7 ms, and the average was 13.4 + 3.8 ms, whereas the average in the AHI (Figure 3A, b graph) was 2.5 + 0.9 ms, and some were as fast as 0.5 ms. Rise times also differed similarly in distribution (Figure 3, a graphs). lntraneuronal decay versus rise time constants for both cells had a correlation coefficient of r < 0.3 (Figure 3, c panels). Frequency histograms of peak amplitudes were constructed from the same series of TTX-insensitive sEPSCs from each neuron. The distributions of peak amplitudes from AHls and SMCs (Figures 4A and 4B, a graphs) were similar, being skewed toward large values. Peak amplitudes versus decay time constants were not correlated (r < 0.1, Figures 4A and 4B, b panels). Cumulative averages of peak amplitudes were 39 k 25 pA for all AHls tested and 30 k 17 pA for all SMCs. We investigated the voltage dependence of sEPSCs in 5 AHls and 6 SMCs. sEPSCs reverted at 1.5 + 4.4 mV and 0.9 + 3.1 mV, respectively. Decay time constants of sEPSCs were not voltage dependent in the range of -90 to -30 mV. However, at positive holding potentials, a voltage-dependent slowing of the decay was observed in both neuronal classes. Perfusion with the NMDA antagonist CPP returned the synaptic current decays to their original values. The current to voltage relationship was similarly linear in the two cell types. However, a correct assessment of linearity was not possibleduetothelargefluctuationof both spontaneous and evoked (see next section) synaptic currents at each holding voltage. lnterneuronal rise times versus decay time constants were compared within the populations of 55 SMCs and 48 AHls (Figure 5). These parameters were

Figure 3. Rise Time and Decay Time Constant Frequency Histograms from 350 Consecutive sEPSCs of an AHI and an SMC and Comparison of lntraneuronal Rise versus Decay Times (A) (a) Frequency histogram of sEPSC rise times from theAHI. Risetimes ranged from 0.1-1.4 ms and averaged 0.4 f 0.22 ms. (b) Frequency histogram of sEPSCdecay times from theAHI. Decaytimeconstants ranged from 0.5-10.2 ms and averaged 2.5 + 0.91 ms. (c) Graph of sEPSC rise times versus decay time constants in the AHI demonstrates poor correlation between these parameters (r = 0.28). (B) (a) Frequency histogram of sEPSC rise times from the SMC. Rise times ranged from 0.3-3.9 ms and averaged 1.7 f 0.83 ms. (b) Frequency histogram of sEPSC decay times from the SMC. Decay time constants ranged from 4.7-31.8 ms and averaged 13.0 + 3.8 ms. (c)Graph of sEPSC peak amplitudes versus decay time constants in the SMC demonstrates poor correlation

Decay Time (msec)

(r = 0.24).

weakly correlated in AHls (r = 0.7) and poorly correlated in SMCs (r = 0.4). Furthermore, membrane time constants (rm) and total electrotonic lengths (L) were similar in AHls (rm = 46 f 15, L = 1.1 + 0.3, n = 14) and SMCs (rm = 51 f 17, L = 0.9 f 0.2, n = 15). Neither of these parameters correlated with the decay time constants in AHls (rm, r = 0.4; L, r = 0.4; n = 14) or SMCs (rm, r = 0.1; L, r = 0.2; n = 15). It has been demonstrated that the focal application ofhighconcentrationsofsucrosewill inducetheasyn-

chronous release of individual synaptic vesicles containing glutamate at restricted synaptic locations (Fatt and Katz, 1952; Bekkers and Stevens, 1989). Therefore, sucrose (300 mM) was briefly pressure applied with a large patch pipette directly onto the somatic and proximal dendritic region of SMCs (n = 4; Figure 6). Prior to sucrose application, sEPSCs occurred at a frequency of 15 + 13 Hz and had a mean rise time of 0.9 + 0.3 ms and a mean decay time constant of 8.9 k 4.5 ms; following local sucrose application, the frequency

Figure 4. sEPSC Peak Amplitude Frequency Histograms Were Skewed toward Large Values in Hilar Neurons (A) (a) Frequency histogram of peak amplitudes of the 350 consecutive sEPSCs from the AHI in Figure 3A and (b) their correlation with the decay time constant derived from the fitting analysis (r = 0.09). The average peak amplitude was 49 + 31 pA, and the average decay time constant was 2.5 + 0.91 ms (mean f SD). (B) (a) Frequency histogram of peak amplitudes of the 350 consecutive sEPSCs from the SMC in Figure 3B and (b) their correlation with the decay time constant derived from the fitting analysis (r = 0.097). The average amplitude was 45 f 24 pA, and the average decay time constant was 13 f 3.8 ms (mean f SD).

75 1 (PA)

1 (PA)

a 4 -I

I 0 Decay Tim: (msec)

Decay Ti%

(msec)

28

Synaptic 749

Currents

in Hllar Neurons

a6-

Y

30 pA 60 msec

0.2

0.4

Rise

B

0.6

time

1.o

0.8

Figure 6. Focal Applicationof chronous Release of Vesicles synaptic Terminals

(msec)

(A) Control sEPSCs occurred at a frequency of 9 Hz and had a mean rise time of 1.0 f 0.4 ms and a mean decay time constant of 11 f 4.8 ms. (B)Sucrose(3OOmM)focallyapplied tothesomaregion increased the frequency of vesicular release to 54 Hz, with only minor effects on rise (0.9 * 0.2 ms) and decay (9.9 i- 4.3 ms) times.

20-

j : ; 5 % g 10:

n

15-

I .s

5

rable kinetics tothe sEPSCs recorded in the samecells (2 AHls and 3 SMCs, Table 1). Electrically evoked EPSCs from cell number AHI 3 and SMC 5 are demonstrated in Figure 7, b traces. Amplitudes of the electri-

1

K 1 D

,

00.0

,

0.4 Rise

Figure 5. lnterneuronal Time Constants within

, . .,

0.8 time

1 .2

1 .6

A

(msec)

Comparison of RiseTimes AHI and SMC Populations

versus

mained time

to 65 + 19 Hz, but the mean rise time reslow (0.9 + 0.2 ms), as did the mean decay

constant

(8.7

+_ 4.1

ms).

Evoked and Spontaneous EPSCs Have Similar Properties TTX-sensitive EPSCs were evoked in hilar neurons by chemical (glutamate ionophoresis) or electrical stimulation of dentate gyrus granule neurons (Figure 7). lonophoretic glutamate pulses onto granule cells evoked bursts of EPSCs in 2 AHls and 2 SMCs (Table 1) with rise and decay times comparable to the sEPSCs recorded in the same neurons, as revealed by analysis of 10 evoked and 50 spontaneous events. Examples of these evoked EPSCs from cell number AHI 1 and SMC 1 are in shown Figure 7, a traces. Peak amplitudes of these chemically evoked events were also similar to those of the sEPSCs. Direct rus

electrical

evoked

variable

stimulation

amplitude

within

the

dentate

gy-

EPSCs, also of compa-

IOmsec

b

Decay

(A) Rise times versus decay time constants in 48 AHls (r = 0.7). (B) Rise times versus decay time constants in 55 SMCs (r = 0.4). Average rise and decay times were obtained from at least 50 sEPSCs in each neuron. sEPSCs in AHls (n = 48) were remarkably fast in rise time (0.5 + 0.2 ms) and decay time constant (3.6 * 1.5 ms), but sEPSCs in SMCs (n = 55) were slower, having a rise time of 1.0 * 0.3 ms and a decay time constant of 9.6 + 4.8 ms. The differences in average rise and decay kinetics between cell types were statistically significant (Student’s t test for grouped data, p < 0.01).

increased

SucroseontoSMCs InducedAsynContaining Glutamate from Pre-

1

aw B b “*

It-

Figure 7. Chemical or Electrical Stimulation of Dentate Granule Cells Evoked EPSCs in Hilar Neurons

Cyrus

(A) (a) An ionophoretic pulse (3 ms, 200 nA) of 0.25 M glutamate (pH 9) onto the cell bodies of dentate granule neurons evoked a burst of EPSCs in an AHI. The asterisk denotes the ejection current artifact. An average of 10 individual synaptic currents from several such bursts displayed a decay time constant of 1.7 * 0.32 ms, similar to the value found for 50 spontaneous events in the same neuron of 1.5 f 0.61 ms. (b) Direct electrical stimulation of the dentate gyrus in proximity with the recorded neuron evoked EPSCs. Two superimposed traces demonstrate the variability of amplitude but not of time course. The decay time constant of the evoked EPSCs in this cell was 3.5 f 0.42 ms, similar to that of the sEPSCs recorded from the same cell, 2.9 f 0.91 ms. (B) (a) An ionophoretic pulse of glutamate onto the cell bodies of dentate granule neurons evoked a burst of EPSCs in an SMC. The asterisk denotes the ejection artifact. The decay time constant of averaged evoked EPSCs was 11.0 f 2.0 ms and of sEPSCs was 13.0 * 4.2 ms. (b) Direct electrical stimulation of the dentate gyrus proximal to the recorded neuron evoked EPSCs. Two superimposed traces are shown. The decay time constant of the evoked EPSCs in this cell was 11.0 + 2.2 ms, similar to that of the sEPSCs recorded from the same cell, 9.9 + 3.2 ms. Vertical calibration bar values: 100 pA for (A, a), 60 pA for (A, b), 60 pA for (B, a), and 400 pA for (B, b).

Neuron 750

Table

1. Evoked

Cell # AHI AHI AHI AHI SMC SMC2 SMC SMC SMC

versus

Spontaneous

Evoked

EPSCs

Rise (ms)

EPSCs in AHls

and

SMCs Spontaneous

Decay

(ms)

Amp.

(PA)

Rise (ms)

1 2 3* 4*

0.21 1.6 0.43 0.81

f + t f

0.11 0.94 0.26 0.31

1.7 3.5 3.5 4.0

f * f f

0.52 1.7 0.41 1.9

154 19 91 495

f * f f

54 9 19 172

0.24 f 0.13 0.52 + 0.41 0.53 * 0.21 0.45 + 0.36

‘I

1.7 1.2 0.95 0.72 1.1

+ * * f f

0.38 0.63 0.39 0.41 0.99

12.0 8.7 10.0 8.6 11.0

* f f + *

2.1 1.6 1.8 2.2 2.2

41 57 230 235 496

f f f f +

18 31 54 74 127

1.6 0.91 0.81 0.63 1.1

3* 4* 5*

Summary of rise either ionophoretic layer. Chemically Evoked currents EPSCs from each in thegranulecell currents were in shown in Figure

Decay

0.62 0.83 0.42 0.12 0.52

(ms)

Amp.

(PA)

1.5 3.2 2.9 3.8

f f f ?r

0.62 1.6 0.91 1.2

59 30 38 61

k f + +

26 21 26 22

13.0 9.7 8.2 6.2 9.9

f f + + f

4.2 5.5 4.6 1.5 3.2

13 30 36 53 46

& k f f f

5 16 16 26 33

times, decay time constants, and peak amplitudes from the 4 AHls and 5 SMCs in which EPSCs were evoked through glutamate application (3 ms, 25-300 nA) onto granule cell somas or electrical stimulation within the granule ceil and electrically evoked currents are listed in the left panel, with electrically evoked currents indicated by an asterisk. are represented as the mean f SD of the given parameter from 10 events. Corresponding values from 50 spontaneous neuron are listed in the right panel for comparison. Note that in all neurons tested, chemical or electrical stimulation layerevokedEPSCsofsimilartimecoursetothecorrespondingsEPSCs.Also,peakamplitudesoftheglutamate-evoked the upper range of sEPSC amplitudes of the same neurons. Examples of the evoked currents from AHls 1 and 3 are 7A and from SMCs 1 and 5 in Figure 78.

tally evoked events were orders of magnitude larger than the spontaneous or ionophoretically induced events. As with the sEPSCs, the decay phase of each EPSC evoked by either method of granulecell stimulation was usually fitted by a single exponential. Aniracetam

f k f f +

EPSCs

Effects

on sEPSCs

from

AHls

and

SMCs

Bath perfusion of the nootropic agent aniracetam (1 mM) increased both the amplitude and duration of synaptic currents in hilar neurons (Figure 8). In AHls (n = 6), the amplitude increased by an average of 40% (from 28 f 11 pA to 39 + 13 PA), whereas in SMCs (n = 7) it increased by 14% (from 33 + 9.9 pA to 38 f 12 PA). The decay time constant increased by 103% in AHls (from 3.4 + 0.5 ms to 6.8 k 1.7 ms), but only by 29% in SMCs (9.4 k 2.8 ms to 12.1 + 3.2 ms). Interestingly, the absolute increase in decay time constant was similar between the two neuronal classes (3.4 ms in AHls versus 2.7 ms in SMCs). The rise time also increased slightly in both neuronal populations(AHIs: from 0.5 f 0.05 ms to 0.7 + 0.09 ms [34%]; SMCs: from 1.2 + 0.4 ms to 1.6 + 0.5 ms [24%]). Discussion

Hilar neurons constitute a heterogeneous population of excitatory and inhibitory neurons (Amaral, 1978; Scharfman et al., 1990), which are thought to be an important regulator of hippocampal activity (Scharfman et al., 1990) and to be involved in the pathogenesis of epilepsy (Scharfman and Schwartzkroin, 1990; Sloviter, 1987). Mossy fibers from the dentate gyrus granule cells provide a primary excitatory drive to these neurons (Scharfman et al., 1990) through characteristic synapsesonto both spiny and aspiny hilar neurons (Amaral, 1978; Ribak and Seress, 1983; Ribak et al., 1985).

Hilar neurons werecharacterized electrophysiologically as inhibitory AHls and excitatory SMCs according to the scheme of Scharfman et al. (Scharfman and Schwartzkroin, 1988; Scharfman et al., 1990; Scharfman, 1991). In addition, the morphology of these cells was compared, with particular regard to the presence or the absence of spines and thorny excrescences on SMCs and AHls, respectively, as has been reported (Scharfman et al., 1990; Scharfman, 1991). In their studies, a common feature of hilar neurons was the high frequency occurrence of spontaneous excitatory synaptic potentials. Using the patchclamp technique in brain slices (Edwards et al., 1989), we have described the biophysical and pharmacologi-

Aa

b

0 a

/

30 pA

50 pA 5 msec

3 mrec Figure 8. Aniracetam (1 mM) induced tudes and Decay Time Constants

Increases

in sEPSCAmpli-

Each trace represents an average of 20 events in the absence (A) and the presence (B) of the drug. (a) sEPSCs from an AHI. The decay time constant increased 96%, from 2.3 ms to 4.5 ms. (b) sEPSCs from an SMC. The decay time constant changed from 10.2 ms to 12.6 ms, a 23% increase.

Synaptic 751

Currents

in Hilar Neurons

cal properties of the currents underlying the spontaneous events. Most of these currents likely resulted from spontaneous vesicular release of neurotransmitter at somatic and dendritic synapses, in analogy to the release of acetylcholine at nicotinic synapses at the neuromuscular junction (Fatt and Katz, 1952), since they were similarly insensitive toTTX and MnCh, respectively, blockers of Na+- and Ca*+-mediated APs. Bath application of CNQX, an antagonist of the non-NMDA subtype of glutamate receptor (Honor+ et al., 1988), totally abolished sEPSCs in most neurons when voltage clamped at -60 to -70 mV, whereas CPP, an antagonist of the NMDA-sensitive glutamate receptor subtype (Davies et al., 1986), lacked effect in such conditions. However, CPP-sensitive NMDA components were sometimes present when membrane potential was held more positive than -30 mV and were usually present at positive membrane holding potentials. We also found several noted exceptions of hilar neurons with NMDA receptors participating in the excitatory synaptic input even when clamped at -60 or -70 mV. Thus, under normal conditions, synaptically released glutamate predominantly activates non-NMDA receptors in hilar neurons, but NMDA receptors may be unmasked under special conditions, similar to the finding of a small NMDA component in the mossy fiber input to CA3 pyramidal cells following stimulation of single granule cells (Isaacson and Nicoll, 1990, Sot. Neurosci., abstract). sEPSCs were slower in SMCs than in AHls. At least three mechanisms could account for such differences: dendritic filtering, spine filtering, or different non-NMDA glutamate receptor subtypes. Dendritic Filtering Several lines of evidence suggest that dendritic filteringisnot responsibleforthesEPSC kineticdifferences between SMCs and AHls. First, the fastest sEPSCs in SMCs were significantly slower than the fastest events in AHls. Rise time and decay time constant frequency histograms were constructed for each SMC and AHI in order to analyze not only average sEPSC kinetics, but the entire range from fast to slow events. This was necessary because the differences in average kinetics observed could arise solely from different electrotonic distances of excitatory synapses on AHls and SMCs, with a greater proportion of remote, slower events in SMCs. However, even when the entire population of sEPSCs in each cell is accounted for, the differences in kinetics are still obvious. Rise times also differed in distribution in a similar manner. Second, interneuronal and intraneuronal rise versus decaywas poorly correlated in SMCs (as was intraneuronal riseversusdecayin AHls). lntraneuronal rise versus decay was not correlated in most neurons tested (r was typically 0.2-0.3), suggesting that dendritic filtering did not dramatically affect decay time constants. However, dendritic filtering may have played a minor role intraneuronally, since in about 10% of both AHls

and SMCs, there was some correlation (r from 0.50.7). In fact, Nelson et al. (1986) estimated an order of magnitude difference in the decay time constant over one length constant in spinal cord neurons. One might propose that most excitatory inputs to AHls occur on the soma, whereas very few inputs to SMCs are proximally located (although this contradicts anatomical evidence[Amaral, 19781). If such were the case, one would expect a greater degree of dendritic filtering in SMCs and strong correlation of interneuronal rise versus decay within the population of SMCs, but not within the AHI population. In fact, the finding that interneuronal rise versus decay times were better correlated in AHls than SMCs suggests that dendritic filtering may be more prominent in AHIs. Third, membrane time constants and electrotonic length were similar in the two classes, and these parameters did not correlate with rise time or decay time constants. If dendriticfilteringcontributedtotheslow sEPSC decay in SMCs, one might expect different time constants and longer total electrotonic lengths in SMCs versus AHls. However, we found no significant differences in either parameter between cell types. Furthermore, these parameters did not correlate with rise times or decay time constants within the populations. In fact, even SMCs with extremely short total electrotonic lengths demonstrated the characteristically prolonged sEPSCs. Furthermore, these findings suggest (as do those of the previous section and the similar values of access resistance) that the quality of whole-cell recordings was similar in the two neuronal classes. Fourth, rise time versus peak amplitude and decay time constant versus peak amplitude were poorly correlated in SMCs and AHls, demonstrating a lack of selectivity in the slowing of sEPSCs. Average peak amplitudes of the sEPSCs were not significantly different between AHls and SMCs; furthermore, peak amplitude distributions were similarly skewed to the right in the two neuronal classes. The similar skewness suggests, but does not prove, a similar range of synaptic input to these hilar neurons. Interpretation of the similar average amplitudes is more difficult because the spine may be electrically isolated from the somatic (or dendritic) voltage clamp. The density of receptors may thus be underestimated at SMC synapses. Peak amplitudes and decay time constants of sEPSCs were not correlated within AHls or SMCs, further indicating that the observed kinetic properties are common to the entire population of sEPSCs. This also suggests that the broad range and rightward skew of the sEPSC peak amplitude distribution does not result from poor space clamping of the distal dendrites, but from other mechanisms such as differences in the number of active receptor-channel complexes at various postsynaptic densities, as proposed for spontaneous inhibitory postsynaptic currents (Edwards et al., 1990). Fifth, ionophoretic glutamate or electrical stimula-

Neuron 752

tion

of dentate

granule

cells

(which

provide

proximal

mossy fiber synapses) failed to induce rapid EPSCs. Anatomical studies have demonstrated excitatory synapses arising from the mossy fibers of dentate gyrus granule cells (Amaral, 1978) limited to the thorny excrescences of the soma and proximal dendrites of SMCs (Ribak and Seress, 1983; Ribak et al., 1985). Electron microscopy studies indicate that nearly all SOmatic and most dendritic asymmetric (excitatory) synapses occur on spines (Ribak et al., 1985). The mossy fiber collaterals also approach AHls (Amaral, 1978), where they are thought to make both somatic and dendritic synapses (Ribak and Seress, 1983). Therefore, to ensure further that similar distributions of events were examined in the two neuronal classes, evoked EPSCs were generated in AHls and SMCs by stimulating (chemically or electrically) dentate gyrus granule cells. Our finding that, in a given neuron, evoked EPSCs did not differ significantly in rise or decay time kinetics from sEPSCs supports our contention that similar distributions of synaptic events were examined in AHls and SMCs. Sixth, similarly, focal application of high osmolarity sucrose to the somatic region of SMCs failed to produce EPSCswith a rapid time course. Such application obviously increased the frequency of events, but failed to significantly modify their time course. The high osmolarity sucrose solution enhances the probability of vesicle release at specific sites (Fatt and Katz, 1952; Bekkers and Stevens, 1989), and since the pressure application pipette was placed directly over the cell body, these events predominantly arose from proximal locations. Although we cannot exclude eventual leakage to remote sites, the contribution of distal events was minimal, as evidenced by the rapid increase and decrease in frequency of events as the pipette was advanced toward and drawn away from the somatic region, respectively. Given the arguments presented, it seems unlikely that dendritic filtering significantly contributed to the 2-fold difference in rise times and nearly 3-fold difference in decay time constants of sEPSCs between SMCs and AHls.

Spine Filtering Theoretical predictions by Rall and others (Rail and Rinzel, 1971; Rall and Segev, 1988; Segevand Rail, 1988) proposed that in neurons such as SMCs, dendritic spines are capable of altering current waveforms through their passive membrane properties; however, in other spine models, such as one proposed by Koch and Poggio (1983), current kinetics are not expected to change. As pointed out by Kawato and Tsukahara (1984) and implicit in Rail’s model, current waveforms as in AHls would only be significantly slowed to reproduce waveforms similar to those seen in SMCs if the spine stem resistance approached the spine input resistance (i.e., spine stem resistance within about l/l0 of spine input resistance). On the other hand, Koch and Poggio (1983) estimated the spine in-

put resistance to be much larger than the spine stem resistance, suggesting that the input resistancewould be extremely high because of the small area of membrane contained in the spine head. Although such estimates were obtained from anatomical measurements of typical spines, it should be noted that it has not been possible to measure directly either of these parameters electrophysiologically. Spine stem resistance may be influenced by cytoarchitecture and organelles in addition to the typical cytoplasmic resistivity. Furthermore, the thorny excrescences of SMCs consist of a complex arrangement of three to four spines with a larger membrane surface area (and thus higher capacitance and lower input resistance) than the typical spine. Experimental electrophysiological evidence for a spine filtering model has been lacking (Sah et al., 1990), perhaps because of the variability in spine geometries between neuronal classes. Chang (1952) suggested that decreases in synaptic strength may be accomplished by increasing spine stem resistance, and later Rall and Rinzel (1971) concluded that such changes could enable plastic changes in the relative weighting of synapses. The large number of synaptic spines typically present on spiny neurons could provide multiple substrates for postsynaptic plasticity, since our results indicate that their presence or absence may significantly modulate synaptic strength. Dendritic spines might be modified during neuronal activity by Cal+-dependent enzymes such as proteases and protein kinases (Lynch and Baudry, 1984). Through these and possibly other biochemical mechanisms, formation and shortor longterm modification of synaptic spine geometry could modulate synaptic strength and thus might strongly relate to the development of the appropriate CNS circuitry and to learning and memory. Thus, our experimental evidence for slower kinetics in spiny neurons supports the proposal that the development or modification of spines could be responsible for changes observed during development or learning and the consolidation of memory (Desmond and Levy, 1983; Lynch and Baudry, 1984). However, further testing of the model is needed to confirm its validity. One particular finding, the similar peak amplitudes of sEPSCs in the two neuronal classes, suggests that the spine models proposed cannot fully account for the differences observed, i.e., synaptic currents will necessarily decrease in amplitude upon spine filtering. For the spine model to account for the peak amplitudes being similar, one must make further assumptions concerning larger conductances at SMC versus AHI synapses. These conductances as well as the slower decay could arise from voltage-gated channels in the spines (Segev and Rail, 1988). However, such conductances were apparently not involved in the sEPSCs of either neuronal class, as shown by the lack of voltage dependence of the sEPSC decays and the insensitivity of sEPSCs to TTX and MnL+. Thus, one must assume a larger synaptic conductance through non-NMDA receptors in SMCs. Although receptor

Synaptic 753

Currents

in Hllar Neurons

density or number may indeed vary aspiny neurons, additional mechanisms NMDA receptor heterogeneity might pler explanation of our results.

Non-NMDA

Glutamate

in spiny such provide

Receptor-lonophore

versus as nona sim-

inwardly rectifying current-voltage relations (Hume et al., 1991;Verdoorn et al., 1991). However, such properties were not observed in either SMCs or AHls, making it impossible to infer subunit heterogeneity on that basis.

Kinetics

Differences in non-NMDA receptor subunit expression might be operative in changing the time course of EPSCs prior to propagation through the spine. At the neuromuscular junction, nicotinic acetylcholine receptors respond to a very brief exposure of neurotransmitter (which is rapidly hydrolyzed) with a very rapid activation and opening of channels followed by a rapid exponential decay that reflects the mean open time of the receptor-ionophore complex (Fatt and Katz, 1951; Magleby and Stevens, 1972). In the CNS, non-NMDA receptor channels mediate similarly fast EPSCs, while NMDA receptor channels mediate slow EPSCs (Forsythe and Westbrook, 1988). Glutamate is not rapidly degraded and will remain in the synaptic cleft until it is cleared by diffusion, but it is unclear if its presence will outlive the mean open time of the non-NMDA receptor channel and thus influence the EPSC decay. However, since even with prolonged application of glutamate, non-NMDA receptor channels desensitize rapidly(Tanget al., 1989,199l;Trussell and Fischbach, 1989), it appears that properties intrinsic to the receptor limit the decay time constant of these synaptic currents as well. Moreover, even the slower rise time and decay time constant of EPSCs mediated by the NMDA receptor-ionophore complex reflect the intrinsic properties of this similar receptor-ionophore complex (Lester et al., 1990). Since multiple non-NMDA receptor subunits have recently been cloned (Keinanen et al., 1990; Sommer et al., 1990), it is conceivable that channels produced with different combinations of subunits could vary in kinetics and produce those differences observed between AHls and SMCs. In support of the receptor heterogeneity proposal, it was recently demonstrated that outside-out patches obtained from a heterogeneous population of cultured hippocampal neurons differ with respect to non-NMDA receptor desensitization rate (Tang and Shi, 1991, Sot. Neurosci., abstract). In previous experiments, Tang et al. (1991) demonstrated that aniracetam (1 mM) prolonged synaptic currents in wholecell recordings from hippocampal slices or cultured neurons and desensitization rates in outside-out patches by a similar percentage in each case (about 100%). In our experiments, aniracetam had similar effects on AHI sEPSCs, but prolonged those in SMCs by only 29%. This smaller percentage increase in decay times of sEPSCs observed in SMCs could result from differences in aniracetam sensitivity due to receptor heterogeneity (Xiao et al., 1991), but it could also simply reflect a rapid synaptic current (i.e., 3 ms decay) increased by 100% (to6ms)followed byspinefiltering. It should also be noted that certain combinations of non-NMDA receptor subunits produce channels with

Conclusion Our findings indicate a basic physiological difference between the excitatory drive to excitatory versus inhibitory hilar neurons that might also apply to other areas such as the neocortex. Furthermore, dynamic alterations of the kinetics of synaptic currents could significantly modify synaptic strength as well as alter summation properties in the soma and dendrites, particularly if the synaptic currents are electrically isolated from the parent dendrites and soma (Jack et al., 1975). A prediction of an electrically isolated spine model is the slowing of the current flowing through the high spine stem resistance in response to a given synaptic input at the spine head (Rail and Rinzel, 1971). In fact, our results indicate that sEPSC rise and decay times are slower in SMCs compared with AHls. Similar differences are observed in the kinetics of EPSCs evoked in SMCs and AHls by the electrical or chemical stimulation of dentate gyrus granule cells. Similar consideration must be given to the possibility that non-NMDA receptor heterogeneity may alter the kinetics of sEPSCs in various neurons. Such differences might reflect the functional roles played by specific neuronal types. Furthermore, since dendritic spines restrict the diffusion of Cal+ (Mtiller and Connor, 1991), the possibility cannot be ignored that biochemical compartmentalization could help regulate glutamate receptor expression or modification. Further investigation with pharmacological agents that affect synaptic currents at the receptor or spine level will help resolve their respective contributions to sEPSC kinetics. Our results with aniracetam are consistent with the spine filtering model, i.e., filtered synaptic currents will be prolonged by an amount (in milliseconds) similar to the increase in the true synaptic current. However, as reported by Xiao et al. (1991), differential effects of aniracetam on different populations of hippocampal neurons may result from the heterogeneity of the glutamate receptor subtypes present. In conclusion, the results presented should spur additional investigations into dendritic spines, nonNMDA receptor subtype expression in spiny versus aspiny neurons, and the modification of synaptic function through these features. Experimental

Procedures

Brain Slices Slices of hippocampus (200 wm) cut transverse to the longitudinal axis were prepared from 11-26 days postnatal Sprague-Dawley rats as described by Edwards et al. (1989). Our experience revealed that mechanical cleaning of the slice surface was not necessary for obtaining giga seals, but it was enough to apply

moderate positive pressure while advancing through the tissue. However, superficial cells were always used to minimize the series resistance. Hilar neurons were easily located with an upright microscope equipped with differential interferential contrast Nomarski optics (UEM, Zeiss, Germany) and an electrically insulated water immersion 40x objective with a long working distance (2 mm). Solutions and Drugs Experiments were performed at room temperature (22OC-24OC) using an oxygenated extracellular medium composed of 120 mM NaCI, 3.1 mM KCI, 1.25 mM KzHPO+ 26 mM NaHCOp, 5.0 mM dextrose, 1.0 mM MgCI,, 2.0 mM CaCl*, and 500 nM glycine. When recording sEPSCs, the extracellular solution was modified to contain additionally 1 PM TTX (Sigma, St. Louis, MO) and 2 mM MnC&. The solution was maintained at pH 7.4 by bubbling with 5% CO* + 95% Oz. The slice was completely submerged in a total volume of 500 ~1 and was continuously perfused at a rate of 5 ml/min. Picrotoxin (Sigma), CNQX (Tocris, Buckhurst Hill, England), and CPP (locris) were dissolved in extracellular medium, while aniracetam (a gift of Dr. Alan Kozikowski, Mayo Clinic, Jacksonville, FL) was dissolved in dimethyl sulfoxide(final concentration
electrotonic voltage

lengths transients

were estifollowing

themethodsof Rall(l969). Whole-cellcurrenttraceswerefiltered at 1500 Hz (-3 dB, B-pole, low pass Bessel filter, Frequency Devices) and stored in an LSI 11173computer(lNDEC System, Sunny Vale, CA) after digitization (5 kHz) with a Data Translation analog to digital converter. Decay time constants of spontaneous and evoked EPSCs were determined from exponential fitting with the II/73 system by using an entirely automated least squares procedure (see Vicini and Schuetze, 1985, for further details). This method uses a Simplex algorithm (Caceci and Cacheris, 1984) to fit the data to either a single or double exponential equation of the form l(t) = If exp(-t/r ,) + IB exp(-t/r,), where I, and Ii are the amplitudes of the sEPSC fast and slow components, and it and T, are their respective decay time constants. Peak amplitudes were measured at the absolute maximum of the sEPSCs, taking into account the noise of the baseline and noise around the peak. Rise times represent the time elapsed from 20%-80% of the peak amplitude of the response. Statistics Results are expressed as mean * SD. When comparing heterogeneous groups, e.g., when investigating variations in control parameters of sEPSC kinetics and amplitude, Student’s t test for grouped data was employed; p < 0.05 was considered as an indication of statistical significance. Acknowledgments We wish tothank Erminio Costa and Franc0 Conti for comments on the manuscript and Steve Byers for technical assistance with the confocal microscope. This work was supported by National Institutes of Health program project grant # POI-NS-28130. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

September

3, 1991; revised

December

30, 1991.

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