GABAB-Receptor-Mediated Currents in Interneurons of the Dentate-Hilus Border

David D. Mott,1 Qiang Li,2 Maxine M. Okazaki,1 Dennis A. Turner,3,4 and Darrell V. Lewis2,4

Departments of  1Pharmacology,  2Pediatrics (Neurology),  3Surgery (Neurosurgery), and  4Neurobiology, Duke University Medical Center, Durham, North Carolina, 27710


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mott, David D., Qiang Li, Maxine M. Okazaki, Dennis A. Turner, and Darrell V. Lewis. GABAB-Receptor-Mediated Currents in Interneurons of the Dentate-Hilus Border. J. Neurophysiol. 82: 1438-1450, 1999. GABAB-receptor-mediated inhibition was investigated in anatomically identified inhibitory interneurons located at the border between the dentate gyrus granule cell layer and hilus. Biocytin staining was used to visualize the morphology of recorded cells. A molecular layer stimulus evoked a pharmacologically isolated slow inhibitory postsynaptic current (IPSC), recorded with whole cell patch-clamp techniques, in 55 of 63 interneurons. Application of the GABAB receptor antagonists, CGP 35348 (400 µM) or CGP 55845 (1 µM) to a subset of 25 interneurons suppressed the slow IPSC by an amount ranging from 10 to 100%. In 56% of these cells, the slow IPSC was entirely GABAB-receptor-mediated. However, in the remaining interneurons, a component of the slow IPSC was resistant to GABAB antagonists. Subtraction of this antagonist resistant current from the slow IPSC isolated the GABAB component (IPSCB). This IPSCB had a similar onset and peak latency to that recorded from granule cells but a significantly shorter duration. The GABAB agonist, baclofen (10 µM), produced a CGP 55845-sensitive outward current in 19 of 27 interneurons. In the eight cells that lacked a baclofen current, strong or repetitive ML stimulation also failed to evoke an IPSCB, indicating that these cells lacked functional GABAB receptor-activated potassium currents. In cells that expressed a baclofen current, the amplitude of this current was ~50% smaller in interneurons with axons that projected into the granule cell dendritic layer (22.2 ± 5.3 pA; mean ± SE) than in interneurons with axons that projected into or near the granule cell body layer (46.1 ± 10.0 pA). Similarly, the IPSCB amplitude was smaller in interneurons projecting to dendritic (9.4 ± 2.7 pA) than perisomatic regions (34.3 ± 5.1 pA). These findings suggest that GABAB inhibition more strongly regulates interneurons with axons that project into perisomatic than dendritic regions. To determine the functional role of GABAB inhibition, we examined the effect of IPSPB on action potential firing and synaptic excitation of these interneurons. IPSPB and IPSPA both suppressed depolarization-induced neuronal firing. However, unlike IPSPA, suppression of firing by IPSPB could be easily overcome with strong depolarization. IPSPB markedly suppressed N-methyl-D-aspartate but not AMPA EPSPs, suggesting that GABAB inhibition may play a role in regulating slow synaptic excitation of these interneurons. Heterogeneous expression of GABAB currents in hilar border interneurons therefore may provide a mechanism for the differential regulation of excitation of these cells and thereby exert an important role in shaping neuronal activity in the dentate gyrus.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The excitability of granule cells, the principal cells of the dentate gyrus, is regulated by a diverse population of inhibitory interneurons located in the molecular layer (ML), granule cell layer (GCL), and hilus. One region particularly rich in glutamic acid decarboxylase (GAD) positive, presumably inhibitory, interneurons is the border of the GCL and the hilus (Houser and Esclapez 1994; Kosaka et al. 1985; Ribak and Seress 1983; Seress and Ribak 1983; Sloviter and Nilaver 1987), an area referred to by Amaral (1978) as the dentate hilus border of Zone 4 or the D/H border zone. Interneurons located in this area form synaptic contacts with specific regions of the granule cells they innervate (Amaral 1978; Halasy and Somogyi 1993; Han et al. 1993; Lorente de No 1934; Mott et al. 1997; Ramon y Cajal 1968; Soriano and Frotscher 1993). Some cells in this area, such as the pyramidal basket cells, have axons that arborize within or very close to the granule cell layer. In contrast, other interneurons are specialized to innervate more distal dendritic regions of granule cells. Interneurons innervating somatic and dendritic regions of granule cells may have different inhibitory effects (Miles et al. 1996). Although many interneurons in this layer innervate granule cells, recent studies also have identified a subgroup of interneurons that are specialized to contact only other inhibitory cells (Gulyás et al. 1996, Hájos et al. 1996). These connected interneuronal networks may play a role in rhythm generation in the hippocampal formation. Differential modulation of D/H border zone interneurons thus may provide a mechanism to alter the functional state of granule cells (Parra et al. 1998).

The activity of interneurons is regulated by both GABAA- and GABAB-receptor-mediated inhibition. GABAA-receptor-mediated inhibitory postsynaptic potentials and currents (IPSP/Cs) have been examined in interneurons in several different regions of the hippocampal formation (Buckmaster and Schwartzkroin 1995; Hájos and Mody 1997; Lacaille 1991; Lacaille and Schwartzkroin 1988a,b; Lacaille et al. 1987; Misgeld and Frotscher 1986; Morin et al. 1996; Soltesz and Mody 1994; Williams et al. 1994). These IPSP/Cs are thought to contribute a necessary timing mechanism for rhythmic oscillation in interneuronal networks and thereby contribute to the generation of gamma frequency oscillations (20-100 Hz) in principal cells (Wang and Buzsáki 1996; Whittington et al. 1995). In contrast, the role of GABAB-receptor-mediated inhibition in interneurons is less clear. GABAB IPSP/Cs have been reported in hippocampal interneurons (Buckmaster and Schwartzkroin 1995; Khazipov et al. 1995; Lacaille 1991); however, they have not been examined in detail. To understand how dentate-hilar border interneurons are regulated. we first must have a clear picture of the currents that are involved and whether the expression of these currents correlates with morphological properties of the interneurons. Therefore a primary aim of this study was to characterize GABAB-receptor-mediated IPSP/Cs in morphologically identified D/H border zone interneurons.

A second aim of this study was to determine the role of GABAB inhibition in regulating the activity of these interneurons. We previously have shown that activation of presynaptic GABAB autoreceptors on inhibitory terminals could suppress the synaptic release of GABA and thereby disinhibit granule cells (Mott et al. 1990, 1993). However, the possibility exists that GABAB receptors located on the somatodendritic membrane of inhibitory interneurons may inhibit interneurons and thus contribute to the disinhibition of granule cells. Because of its slower time course, GABAB inhibition might be expected to have markedly different effects on the frequency of interneuron firing than does GABAA inhibition.

To address these issues, we used the whole cell patch-clamp technique to record pharmacologically isolated slow IPSCs from visually identified interneurons at the dentate-hilus border. The cells were filled with biocytin, allowing us to reconstruct their axonal and dendritic arbors for anatomic identification. We found marked heterogeneity in the magnitude of GABAB responses in these interneurons that largely correlated with their axonal distribution. In addition, we found that IPSPB inhibits interneuronal firing and powerfully suppresses N-methyl-D-aspartate (NMDA) EPSPs. We suggest that GABAB inhibition modulates the activity of D/H border zone interneurons and thereby plays an important role in regulating neuronal activity in the dentate gyrus.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Slice preparation and recording

Transverse 300-µm brain slices were prepared from halothane-anesthetized male Sprague-Dawley rats (16- to 30-days old) using a Vibraslicer (Campden 752), and the hippocampal formation was gently dissected free of the remainder of the brain slice. Hippocampal slices were incubated in warmed (32-34°C), artificial cerebrospinal fluid (ACSF) containing (in mM): 120 NaCl, 25 NaHCO3, 10 dextrose, 3.3 KCl, 1.23 NaH2PO4, 1.8 CaCl2, and 1.2 MgSO4, bubbled with a 95% O2-5% CO2 gas mixture at pH 7.4. For recording, slices were placed into a small submersion chamber maintained at 32-34°C and held in place by a bent piece of platinum wire resting on the surface of the slice. Transilluminated slices were viewed with an upright Nikon Optophot microscope with a ×40 water immersion objective, Hoffman Modulation Contrast optics and infrared filtering.

Our whole cell voltage-clamp technique has been described in our previous publications (Mott et al. 1993; Xie et al. 1992). Briefly, recordings were made with microelectrodes (3-6 MOmega ) pulled from borosilicate glass capillary tubing (1.5 mm OD, 1.05 mm ID, World Precision Instruments, Sarasota, FL) on a Flaming-Brown microelectrode puller. The intracellular solution contained (in mM): 130 Kgluconate, 7 KCl, 10 HEPES, 2 MgATP, and 0.3 TrisGTP, and pH adjusted to 7.2 with KOH. Biocytin (Sigma Chemical, St. Louis, MO) 0.2-0.4% was added for later visualization of the neuron morphology. Osmolalities were from 260 to 275 mOsm for pipette solutions and 284 mOsm for the ACSF.

When used as the internal anion, gluconate has been reported to interact with some types of potassium channels (Zhang et al. 1994) but not the GABAB receptor-coupled potassium conductance (Lenz et al. 1997). However, to confirm this, we compared IPSCBs in a group of 13 interneurons recorded with our gluconate-containing internal solution to the IPSCBs in a group of 9 interneurons recorded with an internal solution in which gluconate had been replaced with an equimolar concentration of methylsulfate. We found no difference in the slope conductance of IPSCB between these groups of cells (independent t-test, P > 0.87), indicating that gluconate had no specific effect on the GABAB conductance recorded in this study.

Stimuli were 0.1-ms, monophasic, cathodal, rectangular, constant current pulses (10-1,000 µA) delivered through a monopolar tungsten electrode placed in the outer molecular layer. An Axopatch 1D amplifier with filtering at 3 kHz and series resistance compensation and capacitance compensation set at zero was used. Series resistance was monitored during the recording, and cells were discarded if seal breakdown or sealing over was detected. Responses were digitized and recorded on magnetic disks using a Nicolet digital oscilloscope (model 410). In addition, responses were digitized by a Digidata 1200 A-D board (Axon Instruments, Foster City, CA) in a PC-based computer using Strathclyde Electrophysiology Software Whole Cell Program developed and generously provided by John Dempster.

Cells selected for recording had somata located near the hilar border of the GCL, were distinctively larger than granule cells, and exhibited basilar dendrites entering the hilus. On entry into the cells, current injections were performed to record typical trains of action potentials. If the cell did not exhibit the typical deep monophasic spike afterhyperpolarizations seen in interneurons in this area, it was not used (Mott et al. 1997; Scharfman 1992).

Fixation, histochemistry, and reconstruction

Our methods have been described previously (Mott et al. 1997). Briefly, slices were fixed in 4% paraformaldehyde containing 0.1% gluteraldehyde in 0.1 M phosphate buffer, and 75-µm sections were cut on a vibratome. Sections were incubated in Avidin-horseradish peroxidase solution (Vectastain ABC Standard Kit) for 2-4 h, washed, and incubated in phosphate buffer containing 0.05% 3'-3-diaminobenzidine tetrahydrochloride, CoCl2 (0.025%), and NiNH4SO4 (0.02%) for 15 min and then H2O2 (0.1% final concentration) until cells were visible. For neuron reconstruction, cells were traced across all sections using a ×100 oil immersion lens and a three-dimensional neuronal reconstruction system consisting of an automated stage and a high resolution monitor (Neurolucida, Microbrightfield, Colchester, VT).

Recovery of the stained cell was achieved in 52 of 110 interneurons from which electrophysiological data were used in this report. The 58 cells in which morphology was not recovered were classified as putative interneurons based on their visual appearance during recording and electrophysiological characteristics (see preceding text). Of the 52 cells from which anatomy could be recovered, identification of axon morphology was possible in 35. In those interneurons the axons of which were well stained, the distribution of the axons in the dentate could be classified into four patterns previously described in detail by Mott et al. (1997), and the reader is referred to that publication for a more extensive discussion of the interneuron morphology. Interneurons, such as basket and axo-axonic cells, the axons of which arborized primarily in the granule cell layer, were termed GCL cells. Cells with axons that ramified throughout the molecular layer were termed TML cells. Many of these TML cells may represent the pyramidal interneurons described by Soriano and Frotscher (1993). Cells with axons primarily in the inner molecular layer were termed IML cells and most likely correspond to the hilar commissural-associational pathway (HICAP) cells of Han et al. (1993). Finally, cells with axons preferring the outer molecular layer are termed OML cells. These cells resemble the hilar perforant pathway-associated (HIPP) cells of Han et al. (1993).

Electrophysiology

Resting membrane potential for each neuron was determined at the beginning of the experiment by the voltage offset accompanying cell access and rechecked at the end of the experiment by measuring the amplitude of the DC offset produced when the cell access was lost. We have corrected for a junction potential of 10 mV by setting the values reported herein at 10 mV more hyperpolarized than the values read off the amplifier. The input resistance of each neuron was determined in current clamp from the amplitude of the voltage deflection produced by a current injection of small amplitude (10-50 pA).

Interneuron recordings lasted 20-60 min. During these recordings, a gradual run down in the amplitude of synaptic currents was noted but not specifically studied. Despite the presence of ATP and GTP in the pipette solutions, NMDA EPSCs, in particular, ran down with time. In contrast, GABAA IPSCs were clearly less prone to this problem and GABAB IPSCs did not show a consistent decrease. The average change in peak amplitude during the first 30 min of recording, expressed as percent of the initial peak amplitude of the evoked PSC, was (mean ± SE) -76 ± 20% for NMDA EPSCs (n = 10 cells), -19 ± 4.9% (n = 8 cells) for GABAA IPSCs, and -10 ± 7.4% for GABAB IPSCs (n = 8 cells).

Values of experimental measurements are given in the results as means ± SE followed by sample number (n). Statistical significance was determined using the Student's t-test or one-way ANOVA with post hoc Bonferroni tests.

Decay kinetics of the IPSCs were analyzed using curve fitting functions in Strathclyde Electrophysiology Software Whole Cell Program (version 1.1) and in Microcal Origin 5.0 (Microcal Software, Northhampton, MA). For the evoked IPSCs, each trace fitted was an average of three to five evoked responses. Each trace was fitted to both a monoexponential and biexponential decay curve. Several criteria were used to conclude whether a given decay phase was better represented by a biexponential or a monoexponential function. One criterion was the ratio of the residual variance of the fit to the background variance as measured by the software. A second criterion was simple visual inspection and often it was quite evident which equation was the better fit to the data. In many cases, the two time constants derived from the biexponential fit differed very little, i.e., by a factor of <= 2, suggesting to us that the most parsimonious interpretation was a monoexponential decay.

Reversal potentials for the IPSCs were determined from current voltage (I-V) curves of the IPSCs elicited at different holding potentials. The expected reversal potential for potassium currents is -97 mV calculated using the Nernst equation with our extracellular potassium concentration of 3.3 mM and intracellular concentration of 137 mM (Hille 1992).

Drugs

Six,7-dinitroquinoxaline-2,3-dione (DNQX; 20 µM) and D(-)-2-amino-5-phosphonovaleric acid (D-APV) (50 µM) were purchased from Tocris Cookson (Bristol, UK). When NMDA-receptor-mediated currents were studied, 10 µM DNQX was used. Bicuculline methiodide (20 µM) and picrotoxin (75 µM) were purchased from Sigma Chemical . QX 314 was obtained from Astra Pharmaceutical Products, (Worcester, MA). GABAB antagonists CGP 35348 and CGP 55845 and the agonist (±)baclofen were supplied generously by Novartis Pharma AG (Basel). All drugs were bath applied in the perfusion medium. Drugs were washed on until a steady-state effect was observed before any measurements were taken. All drugs were dissolved directly into the ACSF except DNQX, which first was dissolved in dimethyl sulfoxide (DMSO) and then added to the ACSF.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we recorded from 110 interneurons at the border of the hilus and the granule cell layer in the dentate gyrus. The input resistance of these interneurons averaged 242 ± 20 MOmega and the resting potential averaged -66.0 ± 0.7 mV. Their morphological and passive electrotonic parameters have been described previously (Mott et al. 1997). In addition, we also recorded from 18 granule cells for comparison with the interneurons. These granule cells had an average input resistance of 324 ± 38 MOmega and rested at -82.2 ± 1.9 mV, significantly more hyperpolarized than the interneurons (P < 0.001, independent t-test).

Slow IPSCs

The slow IPSC was examined in 63 interneurons. In the absence of antagonists a single stimulus to the ML evoked a large fast EPSC followed in most cells by a slow IPSC. Application of DNQX (20 µM), D-APV (50 µM), bicuculline methiodide (20 µM), and picrotoxin (75 µM) blocked the AMPA, NMDA, and GABAA components of the response, respectively, leaving a slow IPSC in 55 of the 63 interneurons (Fig. 1). Spontaneous slow IPSCs were not observed. In 32 of the 55 cells that exhibited a pharmacologically isolated slow IPSC, we delivered a stimulus which evoked a maximal response. The maximal amplitude of the slow IPSC at a holding potential of -70 mV averaged 22.8 ± 2.6 pA but varied markedly over a range from 1.6 to 56.9 pA. Eight of the 63 interneurons exhibited no detectable slow IPSC despite strong and/or repetitive stimulation and the presence of a robust EPSC in the absence of antagonists. These eight cells appeared electrophysiologically healthy in that they had an input resistance (268 ± 30 MOmega ) similar to the average of all interneurons and hyperpolarized resting potentials (-63.0 ± 1.7 mV). In contrast to interneurons, a slow IPSC was present in all 18 granule cells examined. In 10 of these granule cells, we pharmacologically isolated the slow IPSC and delivered a stimulus that evoked a maximal response. In these cells, the maximal amplitude of the slow IPSC averaged 35.7 ± 6.6 pA (range: 8.8-69.9 pA), significantly larger than that recorded from interneurons (P < 0.05, independent t-test).



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Fig. 1. Slow inhibitory postsynaptic currents (IPSCs) in dentate hilar border interneurons. A: Neurolucida reconstruction of an inner molecular layer (IML) interneuron. This cell had aspinous dendrites that extended both into the molecular layer and hilus and an axon which arborized primarily in the inner molecular layer and granule cell layer. Scale bar, 100 µm. Inset, top: recordings from this IML cell revealed a fast excitatory postsynaptic current (EPSC) and a fast IPSC (down-triangle), followed by a slow IPSC (black-down-triangle ) in response to stimulation of the outer molecular layer (down-arrow ). Amplitude of the EPSC is truncated at the - - -. Holding potential: -70 mV. Bottom: application of antagonists of glutamate and GABAA receptors isolated the slow IPSC. B: reconstruction of an outer molecular layer (OML) interneuron. This cell had dendrites that extended into both the hilus and molecular layer and an axon that arose from the apical dendrite and ramified primarily in the outer molecular layer. Scale bar, 100 µm. Inset: OML stimulus evoked a pharmacologically isolated slow IPSC in this OML cell.

The GABAB antagonist, CGP 35348 (400 µM) was applied to seven interneurons. It reversibly blocked the slow IPSC in four of these cells (Fig. 2A). However, in the remaining three cells, this antagonist only partially reduced the peak current (Fig. 2C). Similar results were obtained with the more potent GABAB receptor antagonist, CGP 55845. Application of a maximal concentration of CGP 55845 (1 µM) in a separate group of eighteen cells blocked the slow IPSC in 10, but 8 were partially resistant to this antagonist (Fig. 2C). Furthermore increasing the concentration of CGP 55845 to 10 µM in three of these cells produced no further reduction in the slow IPSC. In contrast, 1 µM CGP 55845 blocked the slow IPSC in all 10 granule cells tested (Fig. 2D). Thus in contrast to principal cells where the slow IPSC was entirely GABAB-receptor mediated, the slow IPSC in some D/H border zone interneurons appeared to be mediated by both GABAB receptors and receptors insensitive to GABAB antagonists.



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Fig. 2. Sensitivity of the slow IPSC to GABAB receptor antagonists. A, top: in this throughout the molecular layer (TML) cell, 400 µM CGP 35348 completely blocked the slow IPSC evoked by an OML stimulus. On washout of the antagonist the slow IPSC recovered. Bottom: individual slow IPSC amplitudes in this cell are plotted against time. Application of CGP 35348 () suppressed the slow IPSC in a reversible manner. · · · , average slow IPSC amplitude in control. Holding potential: -70 mV. B: Neurolucida reconstruction of the interneuron from which these recordings were taken. This cell had aspinous dendrites that ramified widely in the molecular layer and hilus. Axon of this cell was only partially recovered. However, from this partial reconstruction it was possible to identify this interneuron as a TML cell with an axon that arose from the apical dendrite near the top of the granule cell layer and arborized throughout the molecular layer. Scale bar, 100 µm. C, left: in another TML cell CGP 35348 (400 µM) suppressed the slow IPSC by only ~50%. Bottom: component of the response that was sensitive to the antagonist. Holding potential: -65 mV. Right: these scattergrams indicate the degree of block of the slow IPSCs by GABAB antagonists. Each data point represents 1 cell and indicates the peak amplitude of the slow IPSC in the antagonist expressed as percent of the peak amplitude in control. CGP 35348 (400 µM) effectively blocked the slow IPSC in 4 of 7 cells tested with only partial block in the remaining 3 cells. In each cell, the slow IPSCs returned to almost full amplitude after washout of the antagonist. Similarly, CGP 55845 (1 µM) reduced the slow IPSC in all 18 cells tested. However, 8 of these cells exhibited an antagonist resistant component. Histograms show the mean ± SE for the slow IPSC suppression. D, left: slow IPSC evoked in a granule cell was suppressed completely by CGP 55845 (1 µM). Antagonist sensitive component of the slow IPSC in this cell is shown below. Holding potential: -70 mV. Right: scattergram indicating the degree of block of the slow IPSC by CGP 55845 (1 µM) in granule cells. Note that the antagonist produced >92% suppression in every cell tested (n = 10). Histogram shows the mean and standard error for suppression by the antagonist in these cells.

At a holding potential of -70 mV, the high concentration of glutamate and GABAA receptor antagonists used in this study was sufficient to block all inward currents in every granule cell tested (n = 10). In contrast, a small inward current remained in 36 of 63 interneurons (see Figs. 3B and 8A). Therefore where indicated, this resistant EPSC was subtracted from the waveform to isolate the IPSCB.

Baclofen effects

Baclofen (10 µM) produced an outward current in 19 of 27 interneurons and 5 of 5 granule cells on which it was applied (Fig. 3A). In the 19 interneurons in which baclofen produced a current the amplitude of this current at a holding potential of -70 mV averaged 39.5 ± 6.8 pA. This was slightly smaller than the average amplitude of the baclofen current in granule cells at a holding potential of -70 mV (42.9 ± 14.4 pA). Subsequent application of the GABAB receptor antagonist CGP 55845 (1 µM) fully reversed the effect of baclofen in all cells tested.



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Fig. 3. Effect of baclofen on interneurons. A: reconstruction of a granule cell layer (GCL) interneuron. This pyramidal interneuron had a primary apical dendrite that extended through the molecular layer as well as basilar dendrites that branched extensively in the hilus and ascended to the molecular layer. Axon of this cell ramified primarily within the granule cell layer. Scale bar, 100 µm. Inset: in this GCL cell, recorded in voltage clamp at -70 mV, baclofen (10 µM, ) produced an outward current that was antagonized by CGP 55845 (1 µM, black-square). Downward deflections in the current trace represent the response of the cell to hyperpolarizing voltage steps (5 mV, 200 ms) used to monitor the conductance of the baclofen current. B, top left: superimposed current traces from a TML cell show the slow IPSC (Control) and its partial blockade by CGP 55845 (1 µM). Bottom left: subtraction of the waveform in CGP 55845 from the control waveform shows the CGP 55845-sensitive IPSCB. up-arrow , time of the outer molecular layer stimulus. Note that, although the CGP-55845-sensitive baclofen current in this cell was large (see Fig. 5B), the GABAB component of the slow IPSC was quite small. Holding potential: -65 mV. Right: reconstruction of the interneuron from which these responses were recorded. This cell had aspinous dendrites in both the hilus and molecular layer. Axon of this cell was only partially recovered but was sufficient to identify it as a TML cell. Scale bar, 100 µm.

The eight interneurons that did not exhibit a baclofen current had high-input resistances (306.5 ± 65.2 MOmega ) and hyperpolarized membrane potentials (-61.0 ± 2.1 mV), suggesting that they were electrophysiologically healthy. In five of these cells, we were unable to evoke a slow IPSC (see preceding text), suggesting that these interneurons did not possess GABAB-receptor-activated potassium currents. In contrast, the remaining three cells did exhibit a slow IPSC. Although this IPSC was suppressed by baclofen, it recovered in the presence of CGP 55845 (1 µM), suggesting that it was not GABAB-receptor mediated. Thus these findings suggest the presence of a subgroup of interneurons in the dentate hilar border region that lack GABAB-receptor-activated potassium currents.

Application of baclofen to both interneurons and granule cells consistently resulted in the disappearance of IPSCB, presumably due to a reduction of GABA release or to saturation of the GABAB response by baclofen. Interestingly, one interneuron in which the large baclofen current was antagonized completely by CGP 55845 also had a prominent slow IPSC that was only minimally sensitive to CGP 55845 (Fig. 3B). This disparity in the ability of CGP 55845 to antagonize the baclofen current and the IPSC suggests that CGP-55845-sensitive GABAB receptors were present on the postsynaptic membrane of this interneuron but were only minimally activated by synaptically released GABA.

IPSCB heterogeneity

In 13 interneurons that were treated with GABAB antagonists, we recovered sufficient biocytin staining to compare the maximal amplitude of IPSCB with the morphology of the cell. The amplitude of the GABAB component of the slow IPSC in each of these cells was determined by subtracting the antagonist-treated waveform from the control waveform. The maximal amplitude of the IPSCB did not correlate with somatic shape or dendritic morphology but did correlate with axonal distribution. The IPSCB in interneurons with axons which projected to the inner molecular layer (IML cells) and granule cell layer (GCL cells) was similar in amplitude to that recorded from granule cells (Fig. 4A). In contrast, the IPSCB in interneurons with axons projecting to the outer molecular layer (OML cells) and the total molecular layer (TML cells) was significantly smaller. The difference in IPSCB amplitude between these groups of interneurons could not be explained by differences in their input resistance, as the input resistance of TML/OML cells (204.0 ± 25.5 MOmega ) was similar to that of GCL/IML cells (171.0 ± 24.2 MOmega ). In contrast to IPSCB, the amplitude of the antagonist-resistant component of the slow IPSC was not different in interneurons with axons projecting to perisomatic (10.3 ± 3.4 pA, n = 3) or dendritic (10.2 ± 5.0 pA, n = 3) regions of granule cells.



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Fig. 4. GABAB currents are larger in interneurons with axons projecting to perisomatic than dendritic regions of granule cells. A: IPSCB amplitude in interneurons with axons which projected to the dendrites (TML/OML cells, n = 9) was significantly smaller than in interneurons with axons that projected to the cell body region (GCL/IML cells, n = 4, ** P < 0.01) or in granule cells (n = 10, # P < 0.05, ANOVA with post hoc Bonferroni test). B: baclofen produced a current of similar amplitude in granule cells (n = 5) and GCL/IML interneurons (n = 5) but a current of only about half this amplitude in TML/OML interneurons (n = 4). Symbols represent the peak current amplitude in each cell, whereas the bars and error bars represent the mean ± SE for each group of cells.

The differences in IPSCB amplitude could be caused by differences in our ability to activate GABAergic input to these cells. Therefore in nine interneurons, we compared the amplitude of the outward current elicited by baclofen with the axonal projection of that cell. The input resistance (195.3 ± 43.9 MOmega ) and resting membrane potential (-55.0 ± 3.1 mV) of GCL/IML cells combined (n = 5) was similar to that of TML/OML cells combined (222.8 ± 50.4 MOmega ; -55.3 ± 1.6 mV, n = 4). However, like our findings with IPSCB, baclofen produced a twofold larger current in GCL/IML cells than in TML/OML cells (Fig. 4B). These findings indicate that interneurons with axons projecting into or near the granule cell body layer express larger postsynaptic GABAB receptor currents than do interneurons with axons projecting to the granule cell dendrites.

Voltage dependence and conductance of IPSCB

In seven cells it was possible to construct complete current-voltage curves from subtracted IPSCBs. In each cell we measured the peak amplitude of the isolated IPSCB over a range of voltages. A regression line was then fit to each of these curves so that the average slope conductance and reversal potential could be determined. To average the curves between cells, peak currents within a given neuron were normalized to the peak current at -70 mV. The normalized measurements from each cell then were averaged together to produce the I-V curves shown in Fig. 5. As these I-V curves show, the IPSCB in both interneurons and granule cells exhibited no rectification over the range of potentials studied (Fig. 5A). The reversal potential of IPSCB in interneurons was -96.7 ± 1.9 mV, identical to the calculated reversal potential of -97 mV for a potassium conductance. This reversal potential also compared well with that of -94.6 ± 2.0 mV for the IPSCB obtained from four granule cells.



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Fig. 5. Voltage dependence of IPSCB. A: mean current voltage relationship for IPSCB in interneurons is similar to that in granule cells and shows no rectification. I-V curve was constructed by normalizing the current at each potential in a given cell to the current at -70 mV in that cell. These normalized I-V curves were then averaged to produce the plot shown here (n = 7). Symbols and error bars represent the mean current ± SE at each potential. Inset: current traces of the IPSCB evoked at holding potentials of -60, -80, -100, and -110 mV in an interneuron are shown. Resistant PSCs have been subtracted from each trace. B: current voltage relationship for the baclofen current in a TML interneuron shows some rectification. I-V relationship was determined by subtracting the current response to a 2-s voltage ramp from -80 to -120 mV in baclofen from the average of that in control and CGP 55845 (1 µM). TML interneuron from which this recording was taken is shown in Fig. 3B.

In six interneurons the voltage dependence of the baclofen current was examined by delivering voltage ramps from -70 to -120 mV in control, in baclofen and after application of CGP 55845. Subtraction of the ramp delivered in baclofen from the average of the ramps delivered in control and CGP 55845 revealed the component of the ramp current produced by baclofen (Fig. 5B). Unlike IPSCB, the baclofen current displayed some inward rectification over the range of membrane potentials tested. The reversal potential of this current averaged -97.9 ± 3.4 mV, similar to that of IPSCB. In comparison, the reversal potential of the baclofen current in granule cells averaged 91.3 ± 1.2 mV (n = 3).

In five of the seven interneurons in which the I-V relationship of IPSCB was examined, a ML stimulus was used that evoked a maximal IPSCB. In these cells, the slope conductance of IPSCB averaged 0.75 ± 0.12 nS (n = 5; range: 0.6-1.35 nS). This value is significantly smaller (P < 0.05, independent t-test) than the IPSCB conductance measured in granule cells (1.31 ± 0.23 nS, n = 3). Similarly, the conductance of the baclofen current, calculated from the amplitude of voltage steps delivered in control, baclofen, and CGP 55845, was smaller in interneurons (1.7 ± 0.3 nS) than in granule cells (2.6 ± 0.6 nS);, however, this difference was not significant.

IPSCB kinetics

IPSCB kinetics were examined in all 25 interneurons to which we applied GABAB antagonists. For each interneuron, the GABAB component of the slow IPSC was isolated by subtracting the antagonist resistant PSCs. The kinetics of these isolated IPSCBs were studied at a holding potentials of -70 mV. GABAB currents exhibited an onset latency ranging from 18 to 50 ms, a peak latency of 114 to 207 ms, and a duration of 452 to 2,110 ms. Although we found no significant difference in the onset or peak latency of IPSCB in interneurons and granule cells, we did find a difference in the decay of the GABAB current (Table 1). The duration of IPSCB in interneurons was significantly shorter than that in granule cells (P < 0.05, independent t-test). In addition, in interneurons IPSCB exhibited a single exponential decay while, in 9 of the 10 granule cells, IPSCB was better fit with a biexponential decay curve. The IPSC in the remaining granule cell was best fit with a single exponential curve with a decay tau  value of 410 ms. Biphasic current decay in granule cells has been reported previously (Otis et al. 1993).


                              
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Table 1. Kinetic properties of IPSCB

Summation of IPSCB during high-frequency stimulation

We examined summation of isolated IPSCBs by delivering 100-Hz stimulus trains with up to 10 stimuli (n = 4). The stimulus intensity was set to evoke an IPSCB of maximal amplitude. To isolate the GABAB component of the response, we repeated the series of stimulus trains in each cell in the presence of CGP 55845 (1 µM) and subtracted these responses from the control responses in that cell. On average, the summed IPSCB plateaued after about seven stimuli at an amplitude that was 3.8-fold larger than that of a single maximal IPSCB in the same cells (Fig. 6). Both the peak latency (164 ± 16%) and the duration (147 ± 13%) of the summed response to 10 stimuli were markedly longer than those of a single response.



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Fig. 6. Summation of IPSCB. A: superimposed current traces showing summation of IPSCB in an interneuron during 100-Hz stimulus trains of a successively greater number of pulses. Number of stimuli used to evoke each response is indicated to the left of that response. B: for each interneuron the area of the summed IPSCB during stimulus trains of increasing duration was determined relative to the area of a single IPSCB in that cell. These values then were averaged and plotted in the graph shown here. · · · , theoretical linear summation of IPSCB, determined by summating the response to a single stimulus. Symbols and error bars represent mean ± SE C: currents in A are compared against the theoretical linear summation of IPSCB. This theoretical response (---) was derived by summation of the fitted curve to the IPSCB evoked by a single stimulus (top). IPSCB was fit using a modified form of the 4th-power exponential kinetic equation of Otis et al. (1993). To facilitate comparison, the theoretical current amplitude for each of these responses has been normalized. Note that the response evoked by 2 stimuli is larger than the theoretical response (left-arrow ).

The total charge carried by the GABAB current was determined from the net area of IPSCB. This measurement revealed that on the second and third responses of the train the charge carried by the summed IPSCB was actually potentiated relative to the theoretical response expected assuming linear summation of the first maximal IPSCB (Fig. 6C). However, beginning with the fourth stimulus, the summed IPSCB rapidly fell below this theoretical response. Thus by the end of the train, the response to the 10th stimulus added little to the total charge flux of the summed IPSCB beyond that evoked by the 7th stimulus.

Inhibition of interneuron firing by IPSPA and IPSPB

We compared the ability of GABAA and GABAB inhibition to inhibit interneuron firing (n = 6). In current clamp and in the presence of 20 µM DNQX and 50 µM D-APV, a monosynaptic IPSP was evoked during a 2-s-long depolarization-induced train of action potentials (Fig. 7). The monosynaptic IPSP consisted of a fast GABAA IPSP and a slow IPSP. We selected cells in which the slow IPSP kinetics were similar to those of IPSPB to ensure that this response was produced primarily by GABAB receptor activation. With small current injections, the firing rate of the interneuron was slow and both IPSPs were able to completely stop action potential discharge. However, as the amplitude of the current injection increased, the firing rate of the interneuron increased and IPSPB, but not IPSPA, became almost completely ineffective. Thus IPSPA powerfully inhibited interneuron firing at all tested membrane potentials, whereas IPSPB produced a hyperpolarizing inhibition that was effective only during weak to moderate depolarizations.



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Fig. 7. Comparison of the ability of IPSPA and IPSPB to inhibit action potential discharge in interneurons. A, left: voltage recordings of the response of an interneuron to 3 different 2-s depolarizing current injections. As the amplitude of the current injection (shown to the left of each trace) was increased, the cell discharged at a higher frequency. Right: monosynaptic IPSP evoked during the depolarization by an OML stimulus (up-arrow ) inhibited interneuron firing. Monosynaptic IPSP (bottom; 20-pA depolarization), consisted of both a fast (down-triangle) and slow (black-down-triangle ) component. Inhibition produced by the slow IPSP was overcome by strong depolarization. B: comparison of the effect of the fast and slow IPSP on the instantaneous firing frequency of the interneuron during the depolarizing current injections. Line is discontinuous to better indicate inhibition produced by the IPSP. Monosynaptic IPSP is superimposed on the graph to indicate the time course of the fast (down-triangle) and slow (black-down-triangle ) IPSP relative to the inhibition of action potential firing. C: instantaneous firing frequency of the interneuron in the absence of the monosynaptic IPSP shows little variation over the course of each of the 3 depolarizing pulses.

IPSPB suppresses NMDA EPSPs

In cells bathed in DNQX (10 µM), BMI (20 µM), and picrotoxin (75 µM), a ML stimulus evoked an NMDA EPSP followed by an IPSPB. D-APV (50 µM) blocked the NMDA EPSP (n = 13) with only a small resistant EPSP remaining in some cells. Application of CGP 35348 (400 µM) blocked the IPSPB and increased the duration of the NMDA EPSP by 5.0 ± 0.8 fold (Fig. 8A). Because of this longer duration, the area of these responses was increased by >2.5-fold (Fig. 8B). Similarly, the latency to peak of the response increased from 48.1 ± 2.8 ms to 68.1 ± 6.0 ms, a significant difference (P < 0.05, paired t -test). Thus when GABAA inhibition is reduced, IPSPB appears to be capable of markedly truncating the NMDA EPSP in these interneurons.



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Fig. 8. Suppression of the N-methyl-D-aspartate (NMDA) EPSP by IPSPB. A: in this interneuron stimulation of the OML evoked a pharmacologically isolated NMDA EPSP followed by an IPSPB recorded in current clamp. Two minutes after the addition of CGP 55845 (1 µM), IPSPB was blocked, causing the NMDA EPSP to widen. Four minutes after adding CGP 55845, the same stimulus evoked a delayed polysynaptic burst riding on a depolarizing slow wave. Addition of 50 µM D(-)-2-amino-5-phosphonovaleric acid (D-APV) blocked the NMDA EPSP except for the small remaining resistant component. Current was injected to depolarize this cell to -60 mV during stimulation. B: comparison of the area of the NMDA EPSP in control and CGP 55845 (1 µM) indicates a significant increase in NMDA charge flux during GABAB receptor blockade (** P < 0.01, paired t-test, n = 5). C: in this interneuron, paired stimuli delivered to the outer molecular layer (up-arrow ) demonstrate paired-pulse depression of the NMDA EPSP by the IPSPB. First stimulus of the pair evoked an NMDA EPSP followed by an IPSPB. Second stimulus, delivered 150 ms later, during the IPSPB elicited an NMDA EPSP that was markedly suppressed. The addition of CGP 35348 (400 µM) blocked the IPSPB, allowing the 2 NMDA EPSPs to summate. Wash of the CGP 35348 reversed the effect. Current was injected to depolarize this cell to -66 mV during stimulation. D: bar graph shows the effect of CGP 35348 (400 µM) on paired pulse depression of the NMDA EPSP in a group of 6 interneurons. Ordinate shows the ratio of the amplitude of the second NMDA EPSP to that of the 1st NMDA EPSP before the application of CGP 35348 (Ctrl), during bathing in the antagonist (CGP 35348) and after wash (Wash). CGP 35348 significantly reduced paired pulse depression of the NMDA EPSP (** P < 0.01, ANOVA with post hoc Bonferroni test). Amplitude of the first NMDA EPSP of each pair was measured as the difference from the membrane potential at the time of the stimulus to the peak depolarization during that EPSP. Amplitude of the 2nd NMDA EPSP of the pair was measured in the same fashion after subtraction of the response to a single stimulus. E: comparison of the effect of IPSPB on NMDA (n = 20) and AMPA EPSPs (n = 3). Paired stimuli 150 ms apart were delivered such that the 2nd EPSP was evoked at the peak of the IPSPB. Only the NMDA EPSP was significantly depressed (** P < 0.01, paired t test). EPSP amplitudes were measured as described above.

To further demonstrate the effect of IPSPB on the NMDA EPSPs, a paired-pulse paradigm was used in which a test stimulus was delivered to the ML 150-200 ms after a conditioning stimulus, near the peak of the IPSPB. The effect of IPSPB on the NMDA EPSP was quantitated by measuring the amplitude of the conditioning EPSP from baseline and the amplitude of the test EPSP in a similar manner after subtraction of the conditioning waveform. The amplitude of the test NMDA EPSP was clearly depressed in 19 of 20 interneurons tested (Fig. 8, C-E). In cells in which CGP 35348 (400 µM) blocked the IPSPB, it also significantly reduced depression of the test EPSP (Fig. 8D). However, blockade of IPSPB did not completely eliminate depression of the NMDA EPSP. The inability of this antagonist to completely block the depression, despite its complete blockade of IPSPB, suggests that factors other than CGP 35348-sensitive GABAB receptors also may contribute to the depression.

A paired-pulse paradigm also was used to evaluate the effect of IPSPB on AMPA EPSPs. AMPA EPSPs were isolated by blockade of NMDA and GABAA responses with D-APV (50 µM), BMI (20 µM), and picrotoxin (75 µM). These EPSPs could be blocked by subsequent application of DNQX (10 µM). As opposed to its effect on the NMDA EPSP, IPSPB had no effect on the AMPA EPSP (Fig. 8E), suggesting that presynaptic depression of glutamate release did not contribute to paired-pulse depression of NMDA EPSPs.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The principal findings of this study were that in most D/H border zone interneurons, ML stimulation evoked IPSCBs similar to those in granule cells; a subpopulation of these interneurons lacked GABAB currents; the amplitude of GABAB currents was larger in interneurons with perisomatic projections than in those with dendritic projections; IPSPBs inhibited depolarization-induced interneuronal firing during weak, but not strong depolarizations; and IPSPBs effectively blocked NMDA but not AMPA EPSPs.

IPSCBs

IPSCBs have been reported in principal cells throughout the hippocampal formation as well as in interneurons in CA1 stratum pyramidale (Lacaille 1991; Sik et al. 1995) and lacunosum-moleculare (Khazipov et al. 1995). However, this current has not been examined in detail in any interneuronal population. In this study, we have examined the kinetics of IPSCBs recorded from D/H border zone interneurons. We found that the onset and peak latency of IPSCB in these interneurons were similar to those observed in granule cells both in this study as well as in previous reports (Hablitz and Thalmann 1987; Mott et al. 1993; Otis et al. 1993; Staley and Mody 1992; Thalmann and Ayala 1982; Xie et al. 1992). However, the decay rate of IPSCB in interneurons and granule cells differed. In interneurons IPSCBs decayed significantly more rapidly than in granule cells and with monoexponential kinetics, whereas in granule cells they decayed with biexponential kinetics. The mechanism underlying this difference is unknown; however, it may reflect diversity in the subunit composition of either the effector potassium channel or the G protein responsible for the GABAB-receptor-mediated current. During rhythmic activity in the dentate network, these differences in the decay of IPSCB in interneurons and granule cells suggest corresponding differences in the frequency range over which the IPSCB will produce inhibition in these two cell types.

As in granule cells (De Koninck and Mody 1997; Otis et al. 1993), IPSCB in interneurons exhibited a nonrectifying current-voltage relation over a range of membrane potentials from -120 to -60 mV. In contrast, over a similar voltage range the baclofen current showed some inward rectification. Inward rectification of the baclofen current also has been reported in CA3 pyramidal cells (Sodickson and Bean 1996). The reason for this difference in the I-V relationship between synaptically evoked currents and baclofen currents is unclear.

The conductance of IPSCB in D/H border zone interneurons was quite small (~0.75 nS), consistent with its proposed function to provide hyperpolarizing inhibition (Misgeld et al. 1989). Given the single channel conductance of 5-11 pS reported for GABAB-linked potassium channels (De Koninck and Mody 1997; Premkumar et al. 1990), this corresponds to the contribution of 70-150 channels to the peak of a single maximal IPSCB. The conductance of IPSCB in both interneurons and granule cells was clearly smaller than that reported in hippocampal pyramidal cells (range 6-19 nS) (Hablitz and Thalmann 1987; Oleskevich and Lacaille 1992; Rovira et al. 1990), suggesting that the functional effects of IPSCB in interneurons may be weaker than in pyramidal cells.

As in principal cells, repetitive stimulation was more effective than single stimuli at activating GABAB receptors on interneurons. This is thought to occur because GABAB receptors are located extrasynaptically and therefore require GABA spillover from the synaptic cleft for their activation (Isaacson et al. 1993; Thompson and Gähwiler 1992). During the second and third pulses of the train, the amplitude of the individual IPSCBs did not run down but instead was potentiated as might be expected if GABA spillover is increased. After these initial responses, the amplitude of individual IPSCBs progressively declined during the train, such that stimuli after the seventh added little to the overall response. This progressive decline in the amplitude of individual responses may reflect GABA depletion, increased GABA uptake, or the activation of presynaptic GABAB receptors capable of suppressing further GABA release (Mott et al. 1993). Taken together, this behavior suggests that GABAB receptors in these interneurons are optimally activated by short bursts of inhibitory afferent activation, such as occurs during rhythmic oscillation in the hippocampal formation (Ranck 1973).

IPSCB heterogeneity

GABAB current amplitude in interneurons exhibited marked heterogeneity. The lack of both a baclofen current and a slow IPSC in some D/H border zone interneurons suggests the existence of a subpopulation of these interneurons that lack functional GABAB-receptor-activated potassium currents. In those cells that expressed a GABAB current, the maximal amplitude of this current correlated with the axonal projection of the interneuron. The heterogeneity in baclofen current amplitude could not be explained by differences in the dendritic surface area, as there is little difference between the total dendritic length of interneurons that project to perisomatic and dendritic regions of granule cells (Mott et al. 1997). Alternately, because of the slow kinetics of IPSCB, the difference in IPSCB amplitude between these groups of interneurons is unlikely to be caused by differences in dendritic filtering. Furthermore we have found previously that the electrotonic length of both TML and OML cells is slightly shorter than that of GCL and IML cells (Mott et al. 1997). Therefore we suggest that interneurons that project to perisomatic regions of granule cells express greater postsynaptic GABAB receptor function than do interneurons which project to dendritic regions.

A number of possible mechanisms may account for postsynaptic differences in GABAB receptor function. First, it is possible that some interneurons may simply lack or contain fewer GABAB receptors. A second possibility is that these interneurons may contain different subtypes of GABAB receptor (Kaupmann et al. 1997) that couple with differing efficiencies to their effector mechanism. Alternatively, the effector potassium channel may be expressed more abundantly in some interneurons or different subtypes may be expressed. A fourth possibility is that a component of the GABAB receptor system may be persistently downregulated in interneurons that project to dendritic regions of granule cells.

A growing body of evidence indicates that distinct groups of interneurons express receptors for different neurotransmitters. Receptors that have been reported to be differentially expressed in subgroups of interneurons include serotonin (Kawa 1994; Morales and Bloom 1997), muscarinic (Behrends and ten Bruggencate 1993), nicotinic (McQuiston and Madison 1999), noradrenergic (Bergles et al. 1996), and metabotropic glutamate receptors (McBain et al. 1994). Similarly, Lambert and Wilson (1993) have reported heterogeneity in presynaptic GABAB receptor function in CA3 interneurons. Other studies have reported the expression of glutamate (McBain and Dingledine 1993) and GABAA receptors (Gao and Fritschy 1994) with different subunit composition in different subpopulations of interneurons. This diversity provides a mechanism by which interneurons with distinct roles in hippocampal computation might be differentially regulated. The heterogeneous expression of GABAB currents in D/H border zone interneurons may provide another means for the differential modulation of these interneurons.

The animals used in this study were between 16- and 30-days old. Seress and Ribak (1990) reported that by day 16 the axonal plexus of basket cells was typically distributed and relatively mature. Therefore it is not clear that the axonal distribution of other dentate interneurons would be expected to change dramatically after day 16. Although the axonal morphology of D/H border zone interneurons may be developmentally mature, it is unknown whether the expression pattern of postsynaptic GABAB receptors in these interneurons is still maturing during the age range of animals used in this study. In thalamic neurons, functional postsynaptic GABAB receptors are present at birth (Warren et al. 1997);however, in hippocampal pyramidal cells. they are not present before the end of the first postnatal week (Gaiarsa et al. 1995). Therefore although we do not feel that it is likely, we cannot rule out the possibility that differences in GABAB function between these interneuronal groups may reflect differences in the developmental time course of GABAB receptors in some interneuron types.

Resistant PSCs

In addition to the differential expression of IPSCB in D/H border zone interneurons, we also saw heterogeneous expression of two unidentified currents. The first of these currents was a slow IPSC that was resistant to GABAB antagonists. It was present in ~44% of D/H border zone interneurons. However, it was not observed in any of the granule cells tested and has not been reported in principal cells (Davies et al. 1993; Jarolimek et al. 1993; Olpe et al. 1993) or L-M interneurons (Khazipov et al. 1995) of the hippocampus. It is not yet known whether this current is present in other interneurons in the hippocampal formation. The receptor mediating these resistant slow IPSCs is not known. It is possible that they are mediated by a subtype of GABAB receptor not sensitive to the antagonists used in this study (Bonanno and Raiteri 1993). Alternatively, they may be produced by activation of a different receptor system. Dentate inhibitory interneurons are contacted by serotonergic afferents from the median raphe (Halasy et al. 1992), raising the possibility that the resistant IPSC is serotonergic. Indeed, serotonin applied to pyramidal cells can evoke a potassium conductance similar to IPSCB (Andrade et al. 1986; Innis et al. 1988). Alternatively, the resistant IPSC could be an opioid-mediated potassium conductance (Madison and Nicoll 1988; Wimpey and Chavkin 1991), although significant release of opioids by a single ML stimulus seems unlikely (Wagner et al. 1990).

The second unidentified current in these interneurons was a synaptically evoked inward current that was not blocked by DNQX and D-APV and was present in ~57% of the interneurons examined. It appeared similar to the spontaneous depolarizing synaptic response recorded from granule cells in the presence of 4-aminopyridine (Forti and Michelson 1997). The presence of each of these currents in a separate subpopulation of D/H border zone interneurons suggests that, like GABAB receptors, they also may provide a means for the differential modulation of certain subtypes of interneurons.

Functional implications

What may be the functional consequences of IPSC/PB in these interneurons? The slow time course and small conductance increase associated with IPSCB suggest that this form of inhibition will play a more subtle role in regulating interneuron function than does IPSCA. In neocortical neurons (Connors et al. 1988), IPSPB has been suggested as a mechanism to reduce background neuronal firing. As in these neocortical neurons (Connors et al. 1988; McCormick 1989), IPSPB in D/H border zone interneurons was able to block action potentials evoked by weak but not strong depolarizations, suggesting that it may serve a similar function in these cells. Strong membrane depolarization is thought to overcome GABAB inhibition because IPSPB relies primarily on its ability to move the membrane potential away from firing threshold to produce inhibition.

In support of this hyperpolarizing mechanism of inhibition, IPSCB in interneurons was able to inhibit excitatory synaptic responses mediated by voltage-dependent NMDA receptors but not those mediated by AMPA receptors. Inhibition of NMDA EPSPs by IPSPB has been reported previously in CA1 pyramidal cells (Morrisett et al. 1991) and in neurons of the amygdala (Huang and Gean 1994). In D/H border zone interneurons, inhibition of NMDA receptors by IPSP/CB may have important consequences for rhythmic bursting, synaptic plasticity, and/or excitotoxicity in the dentate network. Furthermore the ability of IPSCB to suppress NMDA currents may serve an important compensatory role if GABAA inhibition is compromised. These findings indicate that IPSP/CB inhibits the response of these interneurons to slow and/or voltage-dependent excitatory stimuli, such as that mediated by NMDA receptors, while leaving unaffected the response to strong fast voltage-independent excitatory stimuli, such as that mediated by AMPA receptors. Furthermore they suggest that GABAB inhibition may play an important role in shaping the excitatory response of these interneurons and thus may contribute to the GABAB-receptor-mediated depression of inhibition previously reported in dentate granule cells (Mott and Lewis 1991; Mott et al. 1993).

Conclusions

We conclude that GABAB currents in D/H border zone interneurons modulate interneuron firing rates and inhibit slow excitatory currents. The heterogeneous expression of these currents would produce different levels of GABAB inhibition in subpopulations of interneurons and thus permit these cells to match the computational needs of their environment.


    ACKNOWLEDGMENTS

We thank Drs. S. J. Mickel and H.-R. Olpe, Novartis Pharma AG, Basel, for providing CGP 35348 and CGP 55845 and Dr. John Dempster (Strathclyde University) for generously providing data acquisition and analysis software. We also thank Dr. J. Doherty for comments on a draft of this manuscript and K. Mott for expert editorial assistance.

This work was supported by National Institutes of Health Grants DA-06735 (D. V. Lewis) and AG-13165 (D. A. Turner) and a Veterans Affairs Merit Review Award (D. A. Turner).

Present address of D. D. Mott: Dept. of Pharmacology, Emory University School of Medicine, Atlanta, GA, 30322.


    FOOTNOTES

Address for reprint requests: D. V. Lewis, Dept. of Pediatrics (Neurology), Box 3430, Duke University Medical Center, Durham, NC 27710.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 21 September 1998; accepted in final form 19 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society