Interaction of GABAB-Mediated Inhibition With Voltage-Gated Currents of Pyramidal Cells: Computational Mechanism of a Sensory Searchlight

Neil J. Berman and Leonard Maler

Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Berman, Neil J. and Leonard Maler. Interaction of GABAB-mediated inhibition with voltage-gated currents of pyramidal cells: computational mechanism of a sensory searchlight. J. Neurophysiol. 80: 3197-3213, 1998. This study examines, in the in vitro electrosensory lateral line lobe (ELL) slice preparation, mono- and disynaptic inhibition in pyramidal cells evoked by stimulation of the direct descending pathway from nucleus praeminentialis (Pd). The pathway forms the stratum fibrosum (StF) in the ELL and consists of excitatory fibers from Pd stellate cells that make monosynaptic contact with pyramidal cells and disynaptic inhibitory contacts via local interneurons and of GABAergic inhibitory fibers from Pd bipolar cells. Single or tetanic stimulation (physiological rates of 100-200 Hz) of the StF produced excitatory postsynaptic potentials (EPSPs) or compound EPSPs in ELL pyramidal cells. Slow (>600 ms) and fast inhibitory postsynaptic potentials (IPSPs; 5-50 ms) also were evoked. Application of gamma -aminobutyric acid-A (GABAA) antagonists blocked the fast inhibition and dramatically increased the firing rate response to StF tetanic stimuli. GABAA antagonists also increased the amplitude of the slow IPSP. The slow IPSP was reduced by GABAB antagonists. Blockade of excitatory amino acid (EAA) synaptic transmission allowed the monosynaptic bipolar-cell-mediated inhibition to be studied in isolation: EAA antagonists blocked most of the EPSP response to StF stimulation leaving fast and (an increased amplitude) slow IPSP components. The bipolar-cell IPSPs were mediated by GABAA and GABAB receptors as they were sensitive to GABAA and GABAB antagonists. The bipolar-cell IPSPs scaled with stimulation rate (20-400 Hz), reaching a maximum amplitude at 200 Hz. Inhibitory efficacy of bipolar-cell slow IPSPs were tested by their ability to reduce spiking in the face of sustained or brief current pulses. Established spike trains (by sustained injected current) were little affected by the onset of the slow IPSP. Weak brief currents injected during the slow IPSP were strongly inhibited. Strong brief currents could overcome the slow IPSP inhibitory effect. Inhibition was observed to interact with the intrinsic IA-like K+ currents to produce a complex control of cell spiking. Hyperpolarizing inhibition removes inactivation of IA to prevent subsequent inputs from driving the cell to threshold. Established depolarizing inputs, having allowed IA to inactivate, enable the cell to be highly sensitive to further depolarizing input. The term "conditional inhibition" is proposed to describe the general phenomenon where synaptic inhibition interacts with voltage-sensitive intrinsic currents.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Sensory systems typically are described in terms of an ascending hierarchical flow of information from receptors to the highest levels of the neuraxis where signal recognition presumably occurs. Anatomic studies, however, have demonstrated massive descending or feedback pathways in sensory systems (visual: Hollander 1970; auditory: Ostapoff et al. 1990), and some physiological studies suggest that they play an important role in sensory information processing (visual: Sillito 1984; Sillito et al. 1994; auditory: Ebert and Ostwald 1995). In the electrosensory system of gymnotiform fish, anatomic evidence suggests that the first-order processing region, the electrosensory lateral line lobe (ELL; see Berman and Maler 1998b for a description of ELL laminae, pyramidal cells and interneurons), has massive feedback input (Carr and Maler 1986). Feedback to the ELL is mainly excitatory (glutamatergic: Bastian 1993; Berman et al. 1997; Wang and Maler 1994) and terminates in its molecular layer (Maler 1979). There are two anatomically and functionally distinct feedback pathways to the ELL molecular layer. An indirect feedback pathway originates from granule cells of the overlying cerebellum and terminates in the dorsal molecular layer (DML) of the ELL (Sas and Maler 1987); this projection terminates on pyramidal-cell apical dendrites and on several types of GABAergic ELL interneurons as discussed in Berman and Maler (1998a). A direct feedback pathway emanates from stellate cells of the rhombencephalic nucleus praeminentialis dorsalis (Pd; see Fig. 1 for a simplified circuit diagram), which terminates in the ventral molecular layer (VML) of the ELL (Berman et al. 1997). This is a topographic projection that contacts pyramidal-cell apical dendrites and GABAergic interneurons (vml and stellate cells). The direct feedback projection to the ELL runs in a compact fiber bundle, the stratum fibrosum (StF) (Berman et al. 1997). In addition to glutamatergic fibers that terminate in the VML (Wang and Maler 1994), the StF also contains (in its ventral aspect) GABAergic fibers that terminate densely (with a diffuse topography) on the proximal apical dendrites and somata of ELL pyramidal cells (Maler and Mugnaini 1994). This direct inhibitory feedback pathway originates from the GABAergic bipolar cells in the Pd. These cells are the recipient of excitatory collaterals from the same Pd stellate cell axons (Fig. 1, inset) that form the excitatory component of the StF direct feedback pathway (Maler and Mugnaini 1994; Sas and Maler 1983).


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FIG. 1. Schematic of the cell types and pathways in the electrosensory lateral line lobe (ELL) known (Maler 1979; Maler et al. 1981b) to be the target of stratum fibrosum (StF) terminals. Stellate cells in the nucleus praeminentialis (Pd, nucleus indicated by dashed line box) form a descending excitatory pathway to the ELL. Within the ELL this pathway is called the stratum fibrosum (StF). Pd stellate cell StF fibers make excitatory contact on dendrites of the basilar (BP) and nonbasilar (NBP) pyramidal cells and on GABAergic interneurons (GC2, type 2 granule cells; vml, ventral molecular layer cells; S, stellate cells). PD stellate cell StF fibers also make a small number of gap junction contacts with pyramidal cell (but not interneuron) dendrites (Maler et al. 1981b). A direct descending GABAergic input comes from Pd bipolar cells. This input courses in the ventral region of the same lamina as the excitatory StF fibers and contacts pyramidal cell proximal apical dendrites and somata (Maler and Mugnaini 1994). From the circuitry, a stimulating electrode placed in the StF lamina will drive all elements shown in the circuit. Not shown are the GABAergic polymorphic cells, which may receive input from the excitatory StF pathway; their only ipsilateral projection are to type 1 granule cells. Inset: feedback circuit: reciprocal topographic excitatory connections between BP (and NBP) cells and Pd stellate cells. The GABAergic bipolar cells, which receive stellate cell collaterals, project diffusely back to pyramidal cells in the ELL. Filled dot, solid black neurons and fibers = GABAergic/inhibitory synapses and neurons; open dot, gray-shaded neurons and fibers = Glutamatergic/excitatory synapses and neurons; zigzag icon, electrotonic synapse; bar gradient = approximate laminar spread of gamma -aminobutyric acid (GABA) antagonist applications. Laminae borders shown as gray horizontal dashed lines. DML, dorsal molecular layer; VML, ventral molecular layer; PCL, pyramidal cell layer; pl, plexiform layer; GCL, granule cell layer; DNL, deep neuropil layer; DFL, deep fiber layer.

The anatomy (Maler 1979; Maler et al. 1982), transmitters/receptors (Bottai et al. 1997; Maler and Monaghan 1991, 1994; Maler et al. 1981a; Wang and Maler 1994), and in vivo (Bratton and Bastian 1990) and in vitro (Berman et al. 1997) physiology of the direct excitatory feedback projection have been studied in detail. This has led to the hypotheses that this projection is involved is a "searchlight" mechanism (Bratton and Bastian 1990; Maler and Mugnaini 1993, 1994) and that it can adaptively reduce redundant signals (Bastian 1996a,b; Wang and Maler 1997).

Although the anatomy of the direct feedback inhibitory (bipolar cell) projection suggests that it may be a vital component of the putative feedback searchlight, there is no information on its physiology. This report presents our findings on the mechanisms and capabilities of inhibition of ELL pyramidal cells evoked by activation of the direct feedback pathway. Companion studies deal with the disynaptic inhibition evoked when excitatory feedback afferents (DML, VML) activate GABAergic interneurons (Berman and Maler 1998b) and the inhibition evoked by primary afferent activation (Berman and Maler 1998b). These studies may reveal the subtle functional distinctions between inhibition evoked by differing circuitry of a sensory system.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Slices of ELL were prepared according to a modified method of Mathieson and Maler (1988; see Berman and Maler 1998b). Briefly, fish of either sex were anesthetized by immersion in oxygenated water containing 0.2% 3-aminobenzoic acid ethyl ester (MS-222; Sigma provided all chemicals unless otherwise indicated), then respirated with same during surgery. The brain then was exposed by dissection, blocked to ensure a true transverse section of the ELL, removed, and embedded in low-gelling-temperature agarose [5% in artificial cerebrospinal fluid (ACSF); FMC, Rockland, ME; ACSF contained (in mM): 124 NaCl, 3 KCl, 0.75 KH2PO4, 2 CaCl, 2 MgSO4, 24 NaHCO3, and 10 D-glucose). Transverse sections (350 µm) of the ELL were cut by vibratome (Technical Products International, St. Louis, MO) while immersed in chilled ACSF. Each section was transferred to a carbogenated- (95% O2-5% CO2) and chilled-ACSF holding chamber. Slices then were transferred to an interface type slice chamber where they were maintained at room temperature (22°C) and perfused with carbogenated ACSF (0.5-1 ml/min). The total time from removal of the ELL from its oxygenated blood supply to immersion of the first slice in the holding chamber was ~5-7 min. Recording commenced after a 1.5-2 h recovery period.

Stimulation

Lacquer-coated sharpened monopolar tungsten electrodes (50-µm exposed tip) were placed in the StF ~400-500 µm medial to the recording site in the pyramidal-cell layer (PCL) (see Fig. 1). The laminae of the ELL are discerned easily in the slice preparation using surface illumination. The pyramidal layer forms a dark gray stripe just below the StF. The StF myelinated fibers form an opaque light band, distinct from the adjacent gray matter, allowing precise placement of the stimulating electrode relative to the lamina borders. For some experiments, the stimulating electrode was placed so as to preferentially activate the GABAergic fibers of the bipolar cells, which course just ventral to the StF (see Figs. 1 and 2). Square wave pulse (50-100 µs, 1-50 V, or 20-800 µA cathodal, NL102 or SIU unit, Digitimer, Welwyn Garden City, UK) were delivered to the stimulating electrode. Stimulus intensity was adjusted to provide ~30-50% of maximal response amplitude. High stimulation parameters caused direct or antidromic activation of the cells.


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FIG. 2. Response to stimulation of StF depends on electrode position (see schematic in B, based on Fig. 1). A: response of basilar pyramidal cell (identified by biocytin fill) to stimulation of StF with an electrode placed on the ventral border of the StF (see METHODS; site 1 in B). Low stimulus intensities (1.5-2 V) evoked an inhibitory postsynaptic potential (IPSP; right-arrow), which peaked at ~6 ms. With stronger stimulus intensities, an excitatory postsynaptic potential (EPSP) dominated (*), generating spikes and peaks >= 2 latencies (1.1 and 4.2 ms, right-arrow in 7 V example). Spikes and stimulus artifacts are truncated for clarity in this and all subsequent figures. Note the slow hyperpolarization in the response to 8 V. C-E: comparison of the responses of a basilar pyramidal cell (biocytin-filled) to stimulation at site 1 and site 2 (on the dorsal border of the StF) in B. C: stimulation at site 2 evoked EPSPs at all stimulus intensities. Note that there are >= 3 EPSP peaks (*). D: same cell as in C responded with an early IPSP (*) to low-intensity (1-4 V) stimulation at site 1. At higher intensities, an antidromic spike obscures the IPSP. E: same cell as in D, but with current injection (±0.25 nA) to show that the early IPSP (2-V stimulus) temporally overlapped the EPSP (clearly seen in the depolarized trace).

Recording

Intracellular recordings were made in the centromedial segment (CMS) PCL with 2 M potassium acetate-filled electrodes bevelled to 55-120 MOmega DC resistance. For recovery of cells (see Berman and Maler 1998b), some recordings were made with either biocytin (5% in potassium acetate, bevelled to 70 MOmega ) or Lucifer yellow (tips were backfilled by capillary action with a 10% solution in distilled water, then filled with 2 M potassium acetate, bevelled to 120 MOmega ) pipettes. Dye-filled pipettes were bevelled to ~120 MOmega . All experiments were software controlled (PCLAMP 5.5, Axon Instruments, Foster City, CA or A/Dvance, McKellar Designs Vancouver, BC). Electrical activity was amplified (Axoclamp 2A, Axon Instruments), filtered (TM 503, AM502, Tektronix, Wilsonville, OR: 10 kHz fc), digitized (ITC-16, Instrutech, NY: 1.25 to 50 kHz) and analyzed off-line (IGOR PRO 3.0, Wavemetrics, Lake Oswego, OR). For analysis of neuronal spiking, the instantaneous spike rate first was quantized using a digital filter (30-60 ms triangle base) (Paulin 1992). This filter produces a rate estimate that allows averaging across trials and avoids some of the problems introduced by traditional histogram binning (see Paulin 1992).

Drug applications

Antagonists were dissolved in ACSF at the following concentrations: 100 µM SR(-95531) and 70 µM bicuculline (GABAA antagonist, Tocris Cookson, Ballwin, MO; Research Biochemicals International, Natick, MA); 0.05-1 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) [with dimethyl sulfoxide, alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist], 2 µM DL-2-amino-5-phosphovaleric acid (APV) [N-methyl-D-aspartate (NMDA) receptor antagonist, RBI; Tocris Cookson), 250-500 µM (±)-3-(2-carboxypiperazin-4-gl)-propyl-phosphonic acid (CPP) (NMDA receptor antagonist, phaclofen or saclofen 1-3 mM GABAB receptor antagonists, Tocris Cookson). CNQX and CPP or APV were mixed together to block excitatory amino acid (EAA) transmission. Microdroplets were pressure ejected (Neurophore, Medical Systems, Great Neck, NY) via broken-back glass pipette (10-20 µm). Drop size was adjusted so that a single droplet covered an area of the slice surface ~300-400 µm in diameter and were positioned so that the drug spread was observed to span the PCL, StF, and VML as shown in Fig. 1.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

The data presented in this study were derived from intracellular recordings in the PCL of the CMS from 87 cells in vitro, of which 43 were identified as basilar and 26 as nonbasilar cells (electrophysiologically or histologically) (see Berman and Maler 1998b). There was no difference in the response of these two cell types to stimulation of the StF, and the data therefore were pooled. The recorded cells were driven synaptically by stimulation of the StF just dorsal to the PCL (Fig. 1). The characteristics of the response to StF stimulation are presented first, followed by the effects of GABA antagonism and attempts to isolate the bipolar-cell input by blockade of excitatory transmission. The interaction of bipolar-cell input with an intrinsic potassium conductance, termed "conditional inhibition," and its functional consequences, then are described.

Response to stimulation of Stf

Medium-strength stimulation of the StF evoked excitatory responses (EPSPs) in all pyramidal cells. The EPSPs had between one (Fig. 2A, *) and three (Fig. 2C, *) peaks; these probably correspond to electrotonic, non-NMDA, and NMDA EPSP combinations (see Berman et al. 1997). A histogram (not shown) of all peak latencies was multimodal with peaks at 1.3, 4.5, and 6 ms. The mean latency to the first EPSP peak was 2.04 ± 0.21 (SE) ms (n = 49). The responses often included a hyperpolarizing IPSP before or after the EPSP when the cell resting potential was held at about -60 mV.

Although not explicitly mapped, the IPSPs appeared to be more readily evoked at low thresholds when the stimulating electrode was placed near or at the ventral border (the position of the GABAergic bipolar-cell projection) of the StF. In the experiments shown in Fig. 2, the stimulating electrode was placed at the ventral (site 1) or dorsal (site 2) border of the StF. Stimulation of site 1 evoked obvious IPSPs (Fig. 2, A and D). The IPSPs were evoked readily by low stimulation intensities that did not evoke monosynaptic EPSPs (e.g., Fig. 2A, 1.5 V), but the responses were dominated by EPSPs and spikes at higher stimulus intensities. The lower stimulus threshold of the IPSP suggests that the stimulating electrode was closer to the GABAergic bipolar axons in the StF or that these large diameter axons were stimulated more readily. The low-threshold appearance of a short-latency (presumably antidromic) spike also would be expected from such an electrode position, as the pyramidal-cell efferents course through the plexiform layer just ventral to the PCL (Fig. 1, see Berman et al. 1997).

Responses evoked from dorsal StF (site 2) did not generate obvious IPSPs (e.g., Fig. 2C) at rest. In many cases, the EPSP and early IPSPs overlapped and were not easily separable without pharmacological intervention (see next section) or by current injection (Fig. 2E). Depolarizing the cell revealed an IPSP (Fig. 2E) that completely overlapped (in time) the EPSP.

Bipolar-cell inhibition revealed by EAA antagonists

To determine whether stimulation of the ventral region of StF evoked inhibition via an indirect/disynaptic route or by direct activation of the (Pd) bipolar-cell afferents, we pharmacologically blocked EAA receptors in the region surrounding the recording site (n = 39). Microdroplet drug applications were sufficiently large to cover a substantial portion of the CMS. Figure 3, A (inset) and C, illustrates the typical effect of application of these antagonists on the response to a single stimulus. In this cell, most of the EPSP current is due to NMDA receptors (Fig. 3A), with a smaller CNQX-sensitive (AMPA) component. In this and most cells (n = 33), there was a small persistent electrotonic EPSP that was not blocked by either drug (see Berman et al. 1997). However, removal of AMPA and NMDA EPSPs revealed a large (<= 8 mV) IPSP(s) in cells without such an IPSP component or increased its amplitude if present under control conditions (e.g., Fig. 3A, n = 12); this increase affected mostly the first few hundred milliseconds of the IPSP.


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FIG. 3. Effects of N-methyl-D-aspartate (NMDA; APV or CPP) and non-NMDA [6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)] antagonists on the EPSPs and IPSPs evoked by StF stimulation. A, inset: single-pulse stimulation evoked a large prolonged EPSP under control conditions. Application of APV antagonized most of the EPSP, leaving an early peak. Further application of CNQX blocked this early EPSP revealing an IPSP. Stimulus artifact prevented analysis of very short latency events. Slow time base view (main plot) shows a mostly inhibitory (hyperpolarizing compared with mean "resting" potential, - - -) slow (>800 ms) response with a depolarizing component. This depolarizing component was blocked by APV. CNQX had minimal further effect. B: same cell as in A, but showing the responses to tetanic stimulation (200 Hz, 10 pulses). Under control conditions, the APV-sensitive component seen in A crossed resting membrane potential (- - -) and generated spikes in some trials (reduced by averaging process). APV partially blocked the slow depolarization, revealing a long slow hyperpolarization. CNQX blocked the remaining depolarizing component leaving a large slow hyperpolarization that followed immediately after the cessation of the tetanic stimulus. Under CNQX conditions, there were only 1-2 spikes during the stimulus compared with 10 spikes during control condition. C: cell in which StF stimulation evoked a rapid IPSP (right-arrow) followed by an EPSP. EPSP, but not the rapid IPSP, was blocked by CNQX + CPP. D: same cell responded with a large subthreshold summating EPSP to tetanic stimulation. CNQX + CPP completely blocked this compound EPSP, revealing a slow IPSP. C and D, Vrest (- - -) = -70 mV. E: cell with excitatory response to tetanic stimulation (control trace) and large slow IPSP. After CNQX + APV treatment tetanus evokes IPSPs only with slow and short (right-arrow) components.

The IPSP was mainly monotonic (long and slow; e.g., 3A), except for four cells where there was clearly a very rapid IPSP (peaks at 1- to 3.7-ms latency) after EPSP removal (e.g., Fig. 3C, Right-arrow , E). Note in Fig. 3A, the very long EPSP component (control) was removed by APV, leaving a large long (700 ms) IPSP. At this time scale there was little further effect observed after adding CNQX. By using tetanic stimulation (Fig. 3B) in the same cell, the AMPA EPSP contribution was more prominent (see trace after APV). After blockade of this component, all that was left was a long (800 ms) IPSP. The cell in Fig. 3D produced a long IPSP only when EAA input was blocked and tetanic stimulation was used (cells without a slow IPSP at rest did not reveal a slow IPSP when depolarized). In some cases, tetanic stimulation under EAA receptor blockade also revealed the dual component nature of the bipolar-cell IPSP (Fig. 3E). In 23 cells, the slow IPSP peak amplitude was -1.41 ± 1.51 mV before and -3.07 ± 2.81 mV after EAA blockade (P < 0.05). Five of these cells had no slow IPSP under control conditions. In a minority of cases, EAA blockade decreased the slow IPSP amplitude (4 cells) or had no effect (3 cells). The reversal potential of the slow IPSP after EAA blockade was very negative (-90.6 ± 8.2 mV, n = 8; in 6 other cells reversal was not achieved even in the most hyperpolarized cases -102 ± 7.0 mV).

GABAA receptors and the StF response

Stimulation of the StF bundle, as predicted from anatomic studies, results in a mixed EPSP/IPSP response. During recordings from 22 pyramidal cells, the effect of GABAA antagonists (microdroplets spanning StF, PCL, and VML regions) were tested on the response to StF stimulation. All 22 cells showed an increase in EPSP size and duration (e.g., Fig. 4A), both in single and tetanic (Fig. 4, B and C) stimulation protocols (peak increased from 3.1 ± 1.4 to 4.15 ± 1.95 mV, P < 0.01, n = 11; amplitude at 40 ms latency increased from 0.73 ± 0.70 to 2.29 ± 1.29 mV, P < 0.001, n = 10). Note that the increase in EPSP size appears to begin at ~3 ms; the early unaffected PSP component is probably electrotonic (Berman et al. 1997). During tetanic stimulation (Fig. 4B), bicuculline increased the compound EPSP size, evoking more spikes. The short hyperpolarization after the tetanus (Fig. 4B, arrow, control trace) was ~50 ms in duration, reversed at -62 mV, and was mostly blocked by bicuculline (the hyperpolarization did not require spikes during the tetanic stimulus, so was unlikely to be simply an after-train hyperpolarization; data not shown).


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FIG. 4. Effect of GABAA antagonists on the response to StF stimulation. A: response to single-pulse stimulation under control conditions and after bicuculline application. Bicuculline increased the amplitude of the EPSP peak and its slow decay phase. B: in a different cell, tetanic stimulation (200 Hz, 100 ms, triangles) produces typical summating EPSPs with spikes under control conditions (thick trace). The cell was depolarized (0.1 nA) from rest (-67 mV) to reveal a hyperpolarization after the stimulus (arrow). SR increased the response during the stimulus and reduced this hyperpolarization. C: same cell as in A but with tetanic stimulation (200 Hz, 50 ms) and current injection (±0.5 nA) during control (left) and bicuculline (right) conditions. Spike firing rate for rest and depolarized traces are plotted in the graphs below the raw voltage traces. Under control conditions, tetanic stimulation (shaded region) transiently increased the firing rate established by current injection by a further 50 spikes/s. After bicuculline treatment, the firing rate during the stimulus increased by a further 200 spikes·s-1. Dashed lines indicate Vrest.

Analysis of the effects of GABAA antagonism on the tetanic-evoked responses showed that the most marked increase in firing rate occurred during and just after the stimulus. The cell shown in Fig. 4A was tested with tetanic stimulation at different membrane potentials (Fig. 4C). Under control conditions, tetanic stimulation increased the firing rate by 40-50 spikes.s-1, both at rest and when the cell was depolarized. Application of bicuculline increased the amplitude of the compound EPSP evoked during hyperpolarizing current injection (compare -0.5-nA current traces) and enhanced the response to the tetanic stimulus: firing rate increased by ~200 spikes.s-1 during the tetanus, both at rest and when the cell was depolarized. There was a more modest increase in firing rate in the 50 ms after the tetanus (compare 0.5 nA control and bicuculline cases).

A second common (11 of 15 cells) effect of GABAA receptor-blockade was to increase the amplitude of a slow IPSP or produce a slow IPSP in response to StF stimulation where there was none before (from -0.30 ± 2.39 to -3.39 ± 3.73 mV, n = 11, P < 0.05; reversal potential after blockade: less than -90 mV, n = 6). The illustrated cell (Fig. 5) did not show any evidence for a slow IPSP after single (A, inset) or tetanic stimulation. SR application increased the EPSP size and the spike response as expected, but in addition there was now a slow IPSP (>2-s duration, 1.4-mV peak at 670 ms, extrapolated reversal potential less than -100 mV) after the tetanic stimulus (Fig. 5A). This hyperpolarization was a true IPSP and not a spike-train AHP (afterhyperpolarization) because it could reduce current-evoked firing rates to below control levels (Fig. 5B). Injected step current was used to induce spike firing. StF tetanic stimulation (shaded region) during the current-evoked spiking transiently increased the firing rate. After SR treatment, there was a small uniform increase in cell excitability (firing rate preceding the StF tetanus was slightly higher compared with control) and an increase in the transient firing rate change due to the StF stimulation. Despite these increases in excitability, the firing rate during the slow IPSP time window (Fig. 5B, *) was less than control. This indicates that the mechanism responsible for the slow hyperpolarization was able to inhibit current-evoked firing. In 4 of 15 cells, StF tetanic stimulation evoked a slow depolarization when GABAA receptors were blocked (see Berman et al. 1997); these cells did have a slow IPSP under control conditions.


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FIG. 5. GABAA antagonism uncovers a slow IPSP. A: effect of SR-95531 (SR) on a cell that responded with EPSPs only (no obvious IPSPs) to StF stimulation. With single-pulse stimulation (inset, 5-trial averages), SR increased the amplitude of the peak and slow phase of the EPSP. SR also increased the compound EPSP amplitude and spike rate evoked by tetanic stimulation (main plot, 5-trial averages). However, SR also caused a slow IPSP (*) to be evoked by the tetanic stimulus. That this IPSP is truly inhibitory is demonstrated in B. Vrest = -66 mV. B: mean (2 trials) trifiltered (see METHODS) instantaneous spike rate of the cell in A to tetanic stimulation during control () and SR (···) conditions. Inset: raw voltage traces; note the apparent sag (inset, SR condition, *) in spike rate over the time course of the slow IPSP seen in A. Analysis of the spike rates evoked by different current injection intensities (indicated on left of each panel) shows an increase in spike rate during the tetanic stimulus as a result of GABAA antagonism. However, the spike rate during the 200- to 600-ms window (slow IPSP region) is below (*, 0.3 and 0.4 nA cases) control levels. - - -, 0 spikes/s.

GABAB receptors and the StF response

In the experimental conditions above, the time course of the slow IPSP (hundreds of ms) and its very negative reversal potential (less than -90 mV, see preceding section) indicated that it was likely to be mediated in part via GABAB receptors. This was supported by its pharmacology; with EAA transmission intact, saclofen or phaclofen reduced the amplitude of the slow IPSP (n = 6). Figure 6A shows an example where, without drugs (control), tetanic stimulation produced a slow long-lasting IPSP; phaclofen antagonized some of the early part of the IPSP but left the later component (>600 ms) unchanged. In cells pretreated with GABAA antagonists (see preceding text), saclofen/phaclofen then was applied in an attempt to completely block the slow IPSP (n = 5). These experiments were hampered by the oscillations that were induced by applications of GABAA antagonists in quantities sufficient to block all of the early IPSP (Berman and Maler 1998a; Turner et al. 1991). In the few cells (n = 2) where GABAB antagonists could be applied before the onset of oscillations, there appeared to be total blockade of the slow IPSP. Figure 6B shows one such cell that responded with a slow IPSP after SR treatment (control). Saclofen completely blocked this slow IPSP. Hence StF stimulation activates both GABAA and GABAB receptors.


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FIG. 6. Variable effects of GABAB antagonists on slow IPSPs. A: drug-naive cell responded to tetanic StF stimulation (200 Hz, 50 ms) with a compound EPSP and spikes followed by a slow IPSP. Phaclofen partially blocked an early component of the slow IPSP. B: control response obtained in a cell already exposed to SR; the StF tetanus (black-triangle, 100 Hz) produced a large depolarizing wave that triggered spikes followed by a slow IPSP. Addition of saclofen completely blocked the slow IPSP and prolonged the slow depolarizing potential after the tetanus. C: cell in which excitatory amino acid (EAA) transmission had been blocked with CNQX + CPP. Tetanic stimulation (100 Hz, black-triangle) evoked a medium-duration (~200 ms) IPSP (control, CNQX + CPP present). Saclofen partially blocked this IPSP. Further application of SR blocked the remaining IPSP, which was replaced by a depolarizing slow potential. D: another cell in which excitatory amino acid transmission had been blocked with CNQX + CPP: the StF-evoked slow IPSP was partially sensitive to saclofen. SR application again further antagonized the slow IPSP and produced a slow depolarising potential but also revealed a late slow hyperpolarization that is presumably both SR and saclofen insensitive. All traces are averages of between 5 and 10 trials and Vrest between -65 and -75 mV.

BIPOLAR-CELL INPUT. The direct bipolar-cell IPSP contained both GABAA and GABAB components. After removal of EAA transmission (see Fig. 3), StF tetanic stimulation typically evoked larger slow IPSPs. Because disynaptic IPSPs dependent on local interneurons are presumably eliminated, we interpret the remaining IPSPs as due to activation of GABAergic bipolar-cell axons in the StF (Fig. 6, C and D; see DISCUSSION). The negative reversal potential of the bipolar-cell slow IPSP (less than -100 mV, see Bipolar-cell inhibition revealed by EAA antagonists) is consistent with a K+-mediated GABAB IPSP. This would explain why the slow IPSP amplitude increased after EAA antagonism (see Fig. 3). Under control conditions, a variety of channels are open: EAA channels and voltage-dependent inward currents (reversal potential ~0 mV), GABAA receptors (reversal potential about -65 to -70 mV) (Berman and Maler 1998a) and GABAB receptors (reversal potential less than -100 mV) ( Berman and Maler 1998b); hence the final membrane potential represents the sum of all these currents. Whereas after StF ionotropic EAA synaptic transmission was blocked, only the bipolar-cell input remained, and the GABAB-receptor-mediated IPSP, with its far lower reversal potential, dominated the membrane potential after >100 ms.

Applications of saclofen reduced the amplitude of the evoked IPSP in the 100- to 300-ms window after the tetanus (Fig. 6, C and D; n = 4), confirming that GABAB receptors contributed. Further application of SR blocked the IPSP in this time window completely. In the cell in Fig. 6D, this was accompanied by a new late slow hyperpolarization (starting at 200 ms). As SR and bicuculline invariably induce oscillations (Turner et al. 1991, 1996), the origin of the SR and saclofen-insensitive hyperpolarization remains to be determined. The increased depolarization after SR treatment was probably due to the unmasking of electrotonic EPSPs or AMPA or NDMA receptors not reached/affected by CNQX + CPP but that previously were masked by GABAA inhibition. What is clear is that the StF-evoked IPSPs that survive EAA blockade are sensitive to GABAB as well as GABAA antagonists.

Optimal dynamic range for direct feedback inhibition

The anatomy (Maler and Mugnaini 1994) predicts that the Pd bipolar-cell input (i.e., the slow IPSP seen after EAA receptor antagonism) should be highly effective at inhibiting pyramidal cells because it terminates with large boutons on their somata. Because the firing rates of bipolar cells in vivo are not known, we tested a range of stimulation rates (20-400 Hz, n = 2) to assess the optimal firing rate for this pathway. Tetanic stimuli of between 20 and 400 Hz (10 pulses) were applied in the presence of CNQX + CPP. At rates >20 Hz, a large IPSP was evoked that lasted for 500-600 ms (Fig. 7). As the stimulus rate was increased, the IPSP grew larger and peaked earlier. Two measures of the IPSP are plotted in Fig. 7B: the peak amplitude and exponent of increase (single exponential function fitted from last stimulus pulse to IPSP peak); the latter indicated the rate of IPSP onset. Both measures plateaued at ~200 Hz, with the steepest part of the curves going from 20 to 100/150 Hz; this indicates the dynamic range of this pathway. Note the transition between 100 and 150 Hz; the amplitude only changed marginally, but the IPSP onset was faster and peaked earlier (shift of ~50 ms, measured from last pulse). In functional terms, the IPSP amplitude was the same, but small variations in presynaptic firing rate caused large variations in the temporal characteristic of the inhibition.


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FIG. 7. Stimulus frequency dependence of bipolar inhibitory pathway. Slice treated with CNQX + CPP. A: mean (3 trials) responses to StF tetanic stimulation (20-400 Hz, 10 pulses). Peak amplitude and slope (single exponent fit) of the slow IPSP onset is plotted in B. *, peak amplitude latency shift between the 100- and 150-Hz trials (see text). B: stimulus frequency dependence of IPSP peak (square , left axis) and onset slope (bullet , right axis). Change (Delta ) in both parameters is highlighted when stimulus frequency went from 100 to 150 Hz.

Efficacy of bipolar-cell inhibition on pyramidal-cell excitability

In experiments in which the bipolar-cell input was pharmacologically isolated, we examined the effect of the slow IPSPs on current-evoked spiking (n = 12). Although the IPSP amplitude was a good predictor of inhibitory efficacy when the cell was depolarized (see next paragraph and Fig. 10), there were features of this inhibitory efficacy that at first were puzzling.


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FIG. 10. Effect of the slow IPSP on the activation function (f-I curves) of pyramidal cells. Excitatory transmission blocked with CNQX and CPP. A: response of a cell to injected current pulses alone (top, 0.2/0.5 nA, 100-ms duration) or paired with preceding tetanic stimulation (bottom, 10 stimuli, 100 Hz) of stratum fibrosum (StF + 0.2/0.5 nA). Current pulse was timed to coincide with the peak of the slow IPSP. Average data for this cell is shown in B (left). B: slow IPSP inhibitory effect on f-I curves (5-trial average ± SE) in 3 cells. Intracellular records (5-trial average) above each plot show the slow IPSP evoked in each cell (calibration 5 mV, 100 ms). Curves were obtained under control conditions (open circle , current only: no StF stimulation) and when the current pulse coincided with the peak slow IPSP after StF tetanic stimulation (bullet , StF + current) as in A. Mean firing rate over the pulse duration is plotted against current pulse amplitude. Change in firing rate is shown (···) as a percent inhibition of control (right axis) and absolute change in firing rate (bottom plots: inhibited minus control).

From the IPSPs evoked by tetanic stimulation at rest, we anticipated that the tetanic stimulus would produce a strong and long-lasting inhibition of cell firing---of the order of the inhibition seen (judged by membrane potential hyperpolarizations) at rest. The cell in Fig. 8A was subjected to tetanic stimulation of the ventral StF at 200 Hz (10 pulses); as was typical there was a strong compound EPSP (lower thick trace) with a few spikes during the tetanus (shaded region), followed by a long slow (900 ms) IPSP. The cell then was depolarized (+0.2 nA, t = -100 ms) and the stimulus repeated. On depolarization, the cell fired a train of spikes, which then were interrupted by the stimulus, then resumed 250 ms after the stimulus offset. Prestimulus firing rates were reached after ~500 ms, despite the fact that, at rest, the slow IPSP hyperpolarization lasted 900 ms.


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FIG. 8. Comparison of inhibitory efficacy before and after isolation of the slow bipolar-cell IPSP. A, bottom: control responses of a pyramidal cell to StF tetanic stimulation (200 Hz, 50 ms, shaded region) at rest (thick trace) and during current injection (+0.2 nA, thin trace). Top: instantaneous spike rate (not filtered) derived from depolarized trace. Note drop in spike firing rate during and after the stimulus. B: protocol in A repeated after CNQX + CPP treatment. Drop in spike firing rate now is restricted to the duration of the tetanic stimulus, despite the drug-induced increase in slow IPSP amplitude (compare thick traces in A and B). In addition, there is an even a small increase in spike rate (asterisk, top plot) that overlaps in time with the slow IPSP seen at rest.

It may be argued that depolarizing the cell activated a voltage-dependent component of the StF-evoked EPSP (the NMDA receptor component) (see Berman et al. 1997) that counteracts the IPSP. Exposing the same cell to CNQX and CPP showed that the voltage dependence of the NMDA receptor cannot completely account for this result. CNQX and CPP removed almost all traces of excitatory response to StF stimulation (Fig. 8B, thick lower trace), leaving a larger IPSP with an earlier peak than in Fig. 8A (note that the duration of the IPSP is slightly shorter). Again a depolarizing current was used to evoke spiking. However, now the StF stimulus interrupted firing for a very brief period (70 ms) before the cell returned to prestimulus firing rates. The duration of this brief inhibition suggests that it is mediated by the GABAA-receptor component of the bipolar-cell feedback pathway (see Bipolar cell input and Fig. 4, B and C). Despite the even larger slow IPSP (compared with pre-EAA blockade conditions), the firing rate during most of the slow IPSP window matched or even exceeded (Fig. 8B, *) prestimulus levels. Although the loss of disynaptic inhibition mediated by interneurons (EAA blockade would prevent stellate, vml, and GC2 cells from being activated) would reduce the amount of inhibition evoked by StF stimulation, the minimal inhibitory effect of the large slow IPSP on firing was unexpected.

There are two obvious explanations for this puzzling weakness of slow IPSP inhibition (both with and without EAA blockade) when the cell is depolarized. First, the slow IPSP, mostly GABAB (see above), may be voltage sensitive. Second, voltage-dependent synaptic (NMDA receptor) (Berman et al. 1997) and/or intrinsic (e.g., persistent Na+) (Mathieson and Maler 1988; Turner et al. 1994) currents may dominate the membrane potential (Jaeger et al. 1997), allowing high firing rates on depolarization even though the IPSP is activated. To investigate these possibilities, we looked at the slow IPSP efficacy in more detail.

There is some evidence that GABAB-mediated IPSPs in other systems (e.g., cortical pyramidal cells: Scharfman and Sarvey 1988; thalamic relay cells: Soltesz et al. 1989) are voltage sensitive; their amplitude is reduced with membrane depolarization. This effect was evident in the StF-evoked slow IPSP (Fig. 9A); the IPSP amplitude was maximal at rest and diminished with subthreshold depolarizing currents. This is consistent with the first hypothesis above. However, when EAA transmission was blocked (Fig. 9B), there was an apparent increase in slow IPSP amplitude with depolarization, as expected from the GABAB (K+) channel reversal potential, until threshold for spiking was crossed (0.3 nA case).


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FIG. 9. Voltage dependence of bipolar-cell slow IPSP. A: drug-naive cell stimulated (200 Hz, 50 ms) at rest (- - -) and during current injection (top). right-arrow, latency at which IPSP amplitude was measured (averaged during 20 ms) and plotted (see inset) against prestimulus membrane potential. IPSP amplitude decreased with depolarization. B: same protocol in A followed in cell exposed to CNQX + CPP. Tetanic stimulation evokes a slow IPSP. IPSP amplitude (at 50 ms after last stimulus) is plotted against injected current pulse (prestimulus current-voltage plot not linear over current injection range). IPSP amplitude increased with depolarization until firing threshold is exceeded. Note fast active conductances that are activated at depolarized potentials (*).

This apparent discrepancy can be reconciled by considering the voltage-sensitive NMDA-receptor channels activated by StF tetanic stimulation (see Fig. 3, A and B) (see also Berman et al. 1997). At depolarized levels, more NMDA receptor current (and possibly voltage-sensitive intrinsic currents, Fig. 9B) (see Berman et al. 1997; Mathieson and Maler 1988; Turner et al. 1994) is recruited opposing the hyperpolarizing IPSP. Thus the second hypothesis may better explain the results illustrated in Fig. 9. The apparent weak inhibitory effect of the slow IPSP (e.g., Fig. 8B) mainly may be due to mechanisms other than voltage sensitivity of GABAB K+ channels.

Effect of bipolar-cell inhibition on transient inputs

Although the direct feedback IPSP efficacy appears substantially compromised by the presence of depolarizing currents (Figs. 8 and 9), it is not clear what the mechanism is and whether transient inputs, i.e., inputs activated by electroreceptor activation (see Berman and Maler 1998b), would similarly overwhelm the direct feedback inhibition. This was tested by injecting relatively short current pulses into pyramidal cells before tetanic stimulation and during the peak of the direct feedback IPSP. CPP and CNQX first were applied to the slice to isolate the bipolar cell inhibitory input. Because NMDA receptors are blocked the mechanism described in the previous section (recruitment of NMDA receptor currents with depolarization) will not be operative. Eleven cells were tested with current pulses (0.1-1.2 nA, 0.1-nA increments, 60- to 100-ms duration) to obtain an input/output response curve before and during (70-150 ms after the last StF stimulus pulse) the peak of the slow IPSP that follows a tetanic stimulus (100 ms, 100 Hz). In 3 of the 11 cells, the slow IPSP was small (~0.5 mV) and had little effect on spiking. Examples from three of the other eight cells with varying amplitudes of direct feedback IPSP are shown in Fig. 10. To quantify the inhibitory effect, the response to the current pulses are plotted as frequency-current (f-I) curves; firing frequency is the average rate during the current pulse. Inhibition is plotted both as a percent of control and as the absolute change in firing rate. The maximum decreases in firing rate were correlated significantly (r = 0.84, P < 0.005, n = 8) with the amplitude of the slow IPSPs. Inhibition caused the largest change in firing rate in the midrange of currents tested. With strong depolarizing currents, the direct feedback IPSP caused only a small reduction in firing rate (the 2 f-I curves converge) measured either as a percent of control or as an absolute change in firing rate.

The inhibitory effect on spike rate was not caused by simple algebraic threshold shifts; there were subtle changes in the shapes of the f-I curves. Analysis of the spike times within the responses to current pulse gave some insight into how inhibition was altering the firing behavior of the cell. Under control conditions, the cell was at rest and fired a fairly regular train of action potentials (Fig. 11A, 0.2 and 0.5 nA, same experiment as in Fig. 10). However, when the IPSP hyperpolarized the cell, the time to the first spike was considerably delayed (Fig. 11A, StF + 0.2 nA condition); the latencies to subsequent spikes gradually decreased, and the change in firing rate versus time showed a corresponding increase.


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FIG. 11. Spike latencies altered by slow IPSP. A: instantaneous firing rate vs. spike latency during selected f-I curve current pulses (same cell as in Fig. 10A). Response to current pulse (bullet , tsf + 0.5µnA) during IPSP shows a long lag (delay) to the 1st spike with a slowly increasing firing rate for later spikes. Control response (open circle , 0.5 nA; triangle , 0.2 nA) firing rates showed no obvious trend during the 100-ms current pulse nor any long delay to the first spike (see text). B: detailed plot of latencies to occurrence of 1st, 10th, and 20th spike during the f-I curve current pulses under control (open circle ) and inhibited conditions (bullet ). Mean of 5 trials ± SE, same cell as in A.

These results could not be accounted for by either hypothesis one or two (voltage-dependent GABAB or voltage-dependent NMDA) and a third hypothesis thus is required. We propose that this increase in the latency of the first spike might be due to the action of an IA-like current known to be present in ELL pyramidal cells (Mathieson and Maler 1988) (see DISCUSSION). This therefore was explored for a range of currents in Fig. 11B. The 1st, 10th, and 20th spike times are plotted for each current. Under control conditions, spiking occurs with 0.1-nA injected current albeit at a long latency (mean 60 ms); the latency to the first spike stabilizes at <10 ms with 0.2- to 0.3-nA current injection. During the IPSP, higher currents are required to initiate spiking and the mean latency to the first spike at 0.3 nA is 60 ms. However, for the 1st spike, the "during IPSP" curve is not just shifted to the right (as would be expected for simple subtractive inhibition); note the gradual decrease in latency to the first spike as the current is increased (from 0.3 to 0.7 nA) compared with the sudden shift in latency over 0.1 nA for the control curve. The latencies during control and IPSP conditions converge at 0.7 nA, which is similar to the current at which current-evoked spike rate under control conditions converges to that during the IPSP (Fig. 10B).

The control and inhibited curves for the 10-20th spike had similar shapes, suggesting that simple summation of inward and outward currents might adequately explain the effects of the IPSP at this latency. Analysis of single exponential fits to all the spike rate curves showed that the control and inhibited curves have similar exponential fits from the seventh and later spikes on (data not shown). Inspection of the spike times of all eight cells analyzed showed that this effect of delaying spike onset underlay all the f-I curve changes due to the direct feedback inhibition.

If the effects of the direct feedback IPSP were partially due to inactivation of an IA current, then we would expect that substituting a simple hyperpolarizing current for the slow IPSP should produce the same effects on the f-I curves. This was tested by injecting current the amplitude of which was adjusted to hyperpolarize the cell by the same amount as a StF-evoked slow IPSP. Figure 12 shows the data from one such experiment; the inhibition was similar whether the hyperpolarization was caused by an IPSP (Fig. 12, A1 and B1) or injected current (A2 and B2). In three cells tested, the f-I curves obtained during the IPSP- and current-evoked hyperpolarization were indistinguishable. Subtle effects on f-I due to the small IPSP-associated conductance changes (usually maximum of 5-10% of input resistance) were likely to be obscured by the data variance.


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FIG. 12. Comparison between IPSP and current-induced hyperpolarization on activation (f-I) curve shifts in 1 pyramidal cell (CNQX + CPP present). A: intracellular records (mean of 40 trials) of hyperpolarizations produced by StF tetanic stimulation (A1) or -0.16-nA current pulse (A2). Responses to depolarizing currents used to produce f-I curves are blanked for clarity. B, 1 and 2: f-I curves obtained during control conditions (open circle , no current or StF stimulation), during an IPSP (B1, bullet ), or during current-evoked hyperpolarization (B2, bullet ). ···, percent inhibition and lower plots show the change in firing rate in spikes per second.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Anatomic studies (Maler 1979; Maler and Mugnaini 1994) predicted that after StF stimulation, GABAergic IPSPs would be evoked in ELL pyramidal cells via neurons of the ventral molecular layer (vml cells), stellate cells in VML, type 2 granular interneurons (GC2), and directly via Pd bipolar cell fibers. The results of the present in vitro study allow us to characterize the receptors and IPSPs associated with these cell types. Antagonism of GABAA and GABAB receptors, conventional current-clamp recordings from pyramidal cells, and blockade of EAA transmission were used to isolate the bipolar-cell input from that of the ELL interneurons.

A major feature of activation of the direct feedback pathways originating in Pd was inhibition, consistent with anatomic (Maler and Mugnaini 1994) and in vivo studies (Bastian 1993; Shumway and Maler 1989). The sensitivity of StF-evoked inhibition to GABA antagonists (and the previous iontophoretic study: Berman and Maler 1998b) suggests that most, perhaps all, of this inhibition is mediated via GABAA and GABAB receptors similar to those found in mammalian CNS. The following section discusses the characteristics of the intrinsic (VML, stellate, and GC2) and extrinsic (bipolar-cell pathway) inhibition of pyramidal cells.

Source of input to GABAA and GABAB receptors on pyramidal cells

GABAergic (Maler and Mugnaini 1994) vml and stellate cells in the ventral molecular layer receive glutamatergic input (Wang and Maler 1994) from Pd via the StF. Stimulating the StF would drive these cells, which in turn would inhibit pyramidal cells via their GABAergic contacts on pyramidal cell somata and proximal dendrites. GABAergic GC2s also have dendrites that are well placed to receive StF excitatory input in the VML (Maler 1979). In the slice preparation, stimulating these interneurons is unavoidable, therefore the IPSPs recorded during control conditions are likely to be a combination of input from direct and disynaptic inhibitory feedback pathways. Thus in principle, the GABAA and GABAB receptor components of StF-evoked IPSPs might derive from any combination of these afferents. However, as GC2 (Berman and Maler 1998b), stellate and vml (Berman and Maler 1998b) cell-dependent IPSPs are probably mediated solely by GABAA receptors, the bipolar-cell pathway remains the most likely source of GABAB inhibition.

Physiology of the direct bipolar-cell input to pyramidal cells

The direct GABAergic feedback pathway, identified by Maler and Mugnaini (1994), was activated by placing the stimulating electrode in the ventral region of the StF. These responses also contained EPSPs from the direct glutamatergic (Berman et al. 1997; Maler and Mugnaini 1994; Wang and Maler 1994) pathway in the StF and disynaptic IPSPs as discussed earlier. EAA antagonists blocked the excitatory input, leaving a small electrotonic EPSP (Berman et al. 1997) and large IPSP mediated by GABAA and GABAB receptors. Ultrastructural studies (Maler et al. 1981b) of the VML revealed sparse numbers of gap junction contacts between StF derived boutons and pyramidal-cell dendrites, consistent with the small electrotonic EPSPs.

As there is no evidence of gap junction input to GC2, stellate or vml cell dendrites (Maler et al. 1981b), we conclude that the remaining IPSPs (GABAA and GABAB) were due to the activation of the direct feedback projection from Pd bipolar cells to ELL pyramidal cells and that this projection is the only source of GABAB-receptor-mediated IPSPs evoked by descending feedback afferents. In the case of the NBP cell (Berman and Maler 1998b), the bipolar-cell input is the only input activating GABAB receptors; in the case of basilar pyramidal cells, GABAB receptors are associated with both ovoid (Berman and Maler 1998b) and bipolar-cell input. Of the putative bipolar-cell IPSP components, it appears that the GABAB component may, as in other cells, contribute mainly to the late phase of the evoked IPSP. Although the GABAA component is difficult to isolate due to the overlapping EPSP, when such isolation can be achieved, this IPSP was brief.

Other pharmacological experiments suggested, however, that the GABAA component of the bipolar-cell IPSP was >200 ms in duration (tetani evoke a slowly decaying depolarization after GABAA antagonism: Fig. 6). However, GABAA antagonists caused oscillatory swings in pyramidal-cell membrane potential (Turner et al. 1991) that often are entrained by StF stimulation; this confounds determination of the GABAA receptor component time course. In the following discussion, we assume that the GABAA receptor component is relatively brief in duration (<50 ms, as revealed by single-pulse and tetanic stimulation: Fig. 3), as appears to be the case for the GABAA IPSPs of the same source evoked by primary afferent and DML activation (Berman and Maler 1998a,b).

If indeed all GABAB inhibition is monosynaptic in origin, the apparent increase in slow GABAB (saclofen sensitive) IPSP after application of GABAA-antagonists (e.g., Fig. 5) requires explanation. There are at least two explanations: first, GABA "spillover" to GABAB receptors. Increased activity of disinhibited interneurons may release excess amounts of GABA that spills over to extrasynaptic or nearby synaptic GABAB receptors (see Mody et al. 1994). The much higher affinity of GABAB receptors for GABA would facilitate this effect (Sodickson and Bean 1996). Second, disinhibition of bipolar-cell terminals (i.e., blockade of presynaptic GABAA autoinhibition). This requires presynaptic GABAA autoreceptors on bipolar-cell terminals; this has been shown in other CNS GABA terminals (Sur et al. 1995; Vautrin et al. 1994). It is unclear, however, whether these mechanisms have any physiological relevance.

Estimation of optimal firing frequency of bipolar cells

If the purpose of the direct feedback inhibition is to provide scalable control of the pyramidal cell's response to afferent excitatory input, then we hypothesize that their firing rates will range from 0 to 150 Hz in vivo. In this range, the amplitude and onset of the slow IPSP evoked by bipolar-cell input was approximately linearly proportional to stimulation frequency and the IPSP asymptotes >150 Hz (Fig. 7). Hence changes in Pd bipolar-cell firing rate in this range will produce proportional changes in inhibition of ELL pyramidal cells.

The firing rates of bipolar cells in vivo are not yet known. However, the main input to bipolar cells emanates from collaterals of the Pd stellate cells, which project to the ELL via the StF. Furthermore there do not appear to be any inhibitory boutons associated with bipolar-cell somata (Maler and Mugnaini 1994). Because the firing rate of Pd stellate cells to electrosensory stimuli in vivo is high (ranges from 25 to 300 Hz) (Bratton and Bastian 1990), it is likely that bipolar-cell firing rates are also in this range. If so, then in vivo bipolar-cell firing rates span the optimum range for regulating pyramidal-cell inhibition. The magnitude of the inhibition evoked by stimulation of bipolar-cell axons is the most obvious parameter related to its efficacy in reducing pyramidal-cell discharge. It was clear, however, that the latency to peak of the bipolar-cell-dependent IPSP was even more sensitive to stimulus frequency (Fig. 7). The segments of the ELL have been considered frequency filters (Shumway 1989; Turner et al. 1996), and these temporal shifts in the onset of inhibition therefore also might play a role in regulating the temporal dynamics of the response of pyramidal cells to electrosensory input. A better understanding of these issues will only be possible when the response of Pd bipolar cells to electrosensory input has been investigated directly.

Conditional inhibition: nonlinear interaction of inhibition and intrinsic ionic currents

A surprising feature of the slow IPSPs mediated by the bipolar-cell input to pyramidal cells (i.e., after isolation with EAA antagonists) was their relatively poor ability to reduce an established current-evoked spike train (Fig. 8B) despite their long duration and amplitude at rest (700-800 ms, ~5 mV). The data from the short current pulse injection experiments provided insights into why the slow IPSP had little impact on established spike trains. In an integrate-and-fire neuron, one would predict that a slow GABAB-mediated IPSP would simply increase spiking threshold, i.e., shift the f-I curves to the right (Amthor and Grzywacz 1991). The small conductance increase typically associated with GABAB IPSPs would not shunt much of the injected current, rather the hyperpolarization would increase the spiking threshold. However, something more complex was altering the shape of the f-I curves during GABAB inhibition. Before synaptic inhibition, the curves were a convex saturation function, whereas during the IPSP they were sigmoidal---the slope at low currents was shallower, then became steeper before eventually reaching control firing rates at high currents. This subtle effect was due to changes in the temporal pattern of spike firing during the inhibited train. Analysis of spike timing showed that when the cell was hyperpolarized, there was a lag to the first spike in the train, and the spike rate then increased during the train. This pattern of firing is typical of the response of ELL pyramidal cells to depolarizing current injection. Pharmacological experiments have suggested that it is due to the kinetics of IA-type K+ channels (Mathieson and Maler 1988).

Our hypothesis as to the interaction of GABAB-mediated inhibition and voltage-sensitive conductances is summarized in Fig. 13. Classic IA-type channels are voltage-sensitive K+ channels that are inactivated at depolarized membrane potentials (Fig. 13A1) (Connor and Stevens 1971; McCormick 1991). Hyperpolarization removes IA inactivation (Fig. 13A2); subsequent injection of depolarizing current opens IA channels and thus slows the approach to spike threshold (cf. Fig. 13B, 1 with 2). While depolarized, IA returns to the inactivated state (Fig. 13A2). With sufficiently strong depolarization (Fig. 13B3), IA inactivates rapidly, and there is a potent response to continued depolarizing current injection. Such a mechanism may underlie why bipolar-cell IPSPs are effective at inhibiting small injected currents but are ineffective at preventing large currents from producing near-control spike firing rates. The control f-I curves are obtained at rest where IA is relatively small and rapidly inactivates with injected current (Fig. 13C1). By hyperpolarizing, the cell IA deinactivates, which then has a profound effect on modest current injections (Fig. 13C2). However, large injected currents rapidly shift IA into its inactivated state, allowing near-control firing train patterns and firing rates. Note how the current/spike latency curves converge in the experimental (Fig. 11B) and hypothetical cases (Fig. 13C2). When the cell was depolarized, IA presumably was inactivated rapidly and had little effect on spike latency; in this case, the remaining small amount of inhibition was presumably solely due to the algebraic summation of currents. This view of the nature of the GABAB component of the bipolar-cell IPSP is consistent with the fact that voltage dependence of the f-I curve shapes also could be seen when injected current was used to mimic the effect of activation of bipolar-cell synapses.


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FIG. 13. Schematic of conditional inhibition hypothesis. A, 1 and 2: voltage-dependent IA at different membrane potentials. A1: near resting potential, IA is in an inactivated state, therefore gK(IA) is low. A2: when hyperpolarized, IA is deinactivated and closed (gK(IA) is low). Then, when depolarized, the channel can open allowing K+ to flow through the pore (high gK(IA)). If the depolarisation is maintained the channel slowly inactivates (gK(IA) decreases). B, 1 and 2: conditional inhibition exploits IA voltage dependency to nonlinearly quench weak inputs. B1: weak depolarising input (e.g., electrosensory input) will evoke a strong discharge at rest (or if cell is depolarized) because the depolarizing current is unopposed by gK(IA) (IA inactivated). B2: with conditional inhibition, a slow IPSP (sIPSP) hyperpolarizes the cell and deinactivates IA. Now a weak input tries to depolarize the cell, but the IA channel opens. This increases gk, which opposes further depolarization and therefore delays spiking while IA slowly inactivates. B3: strong input, however, inactivates IA more rapidly leading to robust spiking. C, 1 and 2: schematic of effect of conditional inhibition on activation function of a cell. C1: when cell is at rest (no IPSP, IA inactivated; see A1 and B1), weak inputs can evoke a robust response due to the convex shape of activation function (plot of electrosensory input vs. response). C2: when a slow IPSP (sIPSP) deinactivates IA, the activation function is shifted to the right and the shape alters significantly. Now a weak input will evoke a weak response while the response to a strong input is little altered. Magnitude of changes are emphasized for clarity.

GABAA and GABAB channels classically have been described as mediating shunting (divisive) versus subtractive inhibition respectively (see Koch and Poggio 1987). We already have demonstrated that GABAA-receptor-mediated inhibition in the ELL is voltage sensitive (increased efficacy with depolarization) and thus more complex than anticipated (Berman and Maler 1998a). Similarly, for inhibition mediated by GABAB receptors in the ELL, our results suggest that "subtractive" inhibition may be an inadequate term for its complex properties. We therefore propose the term "conditional inhibition" to reflect the complex interplay between GABAB inhibitory input and the voltage-sensitive conductances of the target neuron. The efficacy of the inhibition is conditional on a voltage sensitivity (or nonlinearity) of an ionic conductance(s) in the cell. Conditional inhibition allows inhibition (not just GABAB) to operate in several computationally rich ways.

First, instead of inhibition being simply proportional to presynaptic activity, it is also sensitive to the history of the postsynaptic cell. If the postsynaptic cell is in a moderately excitatory state (e.g., is responding to an optimal electrosensory stimulus), inhibition will be ineffective at changing the cell's state. If the cell is not being excited, then the inhibition will set the cell into a hyporesponsive state; moderate stimuli will not be effective in driving the cell. However, strong stimuli will overcome and remove the intrinsic component of conditional inhibition (IA in the case of ELL pyramidal cells). This has implications for the participation of the bipolar cell pathway in attention (see following text).

Second, the intrinsic component of conditional inhibition provides a mechanism for modulating the cell's activation function (f-I curve) and therefore will determine the response to any excitatory synaptic input. In this case, altering GABAB channels themselves or altering inward (Na+ or NMDA receptor) or outward (IA) currents [via second messengers, PKC (Covarrubias et al. 1994), PKA (Huang et al. 1994), see also MODULATION OF NONLINEARITY, and through phosphorylation by CamKII (Roeper et al. 1997)] might shift the activation function from convex saturation through linear to sigmoida. Recent theoretical studies (Bell and Sejnowski 1997; Koch et al. 1997) have suggested that modifying a neuron's activation function via its voltage-sensitive conductances may be a useful mechanism by which the cell adapts to the mean and variance of its synaptic inputs. Evidence from an invertebrate preparation suggests that the modulation of intrinsic conductances may in fact be a mechanism used in plasticity (Marder et al. 1996). The spatially localized modulation of activation functions may allow for both rapid and precise regulation of spatial and temporal filtering (see following text) in the ELL without altering the synaptic efficacy of primary afferent input. Such a mechanism might perhaps be found in other sensory systems.

Conditional inhibition is likely to be a widespread mechanism for modulating inhibitory efficacy. Getting (1983) describes an invertebrate cell where inhibition removes inactivation of IA, thereby delaying the response to excitation; this is similar to our explanation of the effect of the bipolar-cell IPSP on spike latency. In mammalian neurons with a strong IA, the efficacy of inhibition is greatly enhanced by deinactivation of IA (thalamic relay cells, McCormick 1991; cerebellar Purkinje cells, Midtgaard 1992). The functional significance of this interaction in a sensory neuron, however, remains obscure. There have been suggestions that the lag in "lagged" cells of the lateral geniculate nucleus (Heggelund and Hartveit 1990) is due to interactions between IA and inhibition (McCormick 1991). More interesting is the hypothesis that the interaction plays a role in attentional filtering (during sleep and arousal) (McCormick 1991); this may indeed be the functional significance of conditional inhibition in the ELL descending feedback pathway (see following text). A similar complex interplay between GABAB inhibitory input-evoked voltage changes and intrinsic currents other than IA has been reported for thalamic cells (low-threshold calcium) (Crunelli and Leresche 1991; Ulrich and Huguenard 1996). The ELL may be a more amenable preparation for the study of conditional inhibition in sensory processing because ELL pyramidal cells have a strong IA current (Mathieson and Maler 1988) and are only one synapse away from peripheral electroreceptors.

Implications for models of ELL: support for a searchlight function

The direct Pd to ELL projection may act as an attention focusing mechanism (for details, see Berman et al. 1997; Bratton and Bastian 1990; Maler and Mugnaini 1993, 1994), similar to the "searchlight of attention" role proposed for the descending corticothalamic feedback projections in the visual system (Crick 1984). Part of the searchlight requirements are met by this projection; reciprocal topographic excitatory input combined with spatially diffuse inhibitory input: 1) Pd stellate cells provide direct reciprocal and topographic excitatory input to ELL and are highly sensitive to moving targets even at some distance from the fish but adapt rapidly to persistent inputs (Bratton and Bastian 1990). 2) The bipolar cell projection to ELL pyramidal cells is relatively diffuse (Maler and Mugnaini 1994) and bipolar cells are likely to be driven by Pd stellate cell activity (see preceding text).

The remaining requirement is a nonlinear, thresholding mechanism. We have identified at least three ways in which GABAB (or any hyperpolarizing IPSP) could produce nonlinear transforms of excitation.

First, the long duration of the GABAB IPSP makes it well suited to control NMDA inputs: the NMDA receptor component of the Pd-ELL excitatory pathway is voltage dependent and hence can exhibit threshold behavior (Berman et al. 1997). [The shorter-acting bipolar-cell GABAA IPSP may regulate the shorter duration AMPA receptor input (Berman et al. 1997).]

Second, hyperpolarization will reduce voltage-dependent persistent sodium inward currents in pyramidal cells (Berman et al. 1997).

Third, conditional inhibition: the GABAB-receptor-mediated shift of the pyramidal cell's activation function from convex to sigmoidal implies that weak, transient excitation will be very effectively quenched by the combined nonlinear effects of relatively small inward currents (NMDA receptor and Na+), bipolar-cell IPSPs and deinactivated IA. Sustained or strong excitation (i.e., a large moving stimulus) will increase the inward currents, thereby inactivating IA; this will permit these stimuli to break though the inhibition (cross the threshold) and activate the pyramidal cells. Thus conditional inhibition provides a means whereby moderate bipolar-cell activity is highly effective in its putative thresholding role in the sensory searchlight. This implies that the direct inhibitory feedback pathway will be critical in filtering signals from noise.

MODULATION OF NONLINEARITY. The effectiveness and/or dynamics of conditional inhibition may very well be controlled by modulation of the efficacy of GABAB receptors (G protein coupled to ion channels), voltage-sensitive (INaP, IA), or synaptic (NMDA receptor) channels in local populations of pyramidal cells. Conditional inhibition then would vary across, or even within, the ELL segments, affecting segmental or local spatial and temporal filtering. The modulation may occur via second-messenger systems that have been identified in the ELL (IP3: Berman et al. 1995; protein kinase A, C and Ca2+/calmodulin dependent kinase II: Maler, unpublished observations). In other systems, these have been shown to modulate the channels mentioned earlier. Furthermore, control of conditional inhibition through these modulatory mechanisms may underlie Bastian's (1996a) recently demonstrated anti-Hebbian plasticity of ELL pyramidal cells in response to local input paired with redundant global input; the redundant input is nulled out locally.

Effect on frequency filtering

Conditional inhibition may have complex effects on the temporal filtering properties of pyramidal cells. From the data presented, conditional inhibition had a strong effect on spike timing for the first 40-50 ms with moderate excitation. The effect decayed as IA was inactivated. We therefore predict that, when conditional inhibition is active, pyramidal cells act as low-pass filters: brief inputs will be attenuated, whereas sustained inputs that persist for the duration required for IA inactivation will be able to control cell firing. In terms of electrosensory function, slow-moving objects near the fish (producing AM of the electric organ discharge of <10 Hz) will overcome this conditional inhibition, whereas the response to rapidly moving objects or brief communication signals (chirps) (see Zupanc and Maler 1997) will be blocked. The time constants of IA activation and inactivation for ELL pyramidal cells will determine the corner frequency of the sensory filter. Note that the fast IPSPs will not deinactivate IA if the time constant of removal of inactivation is of the order of that found in other cells (91 ms) (McCormick 1991). Thus activation of rapid GABAA inhibition from VML, stellate, or GC2 cells may not interact significantly with IA. Clearly, detailed voltage-clamp studies are required to characterize the dynamics of IA; the time constants and voltage dependency of activation, inactivation, and removal of inactivation. This data then can be used to model realistically the impact of conditional inhibition on sensory filtering.

With the information gained in this study about the mechanisms of inhibition used by the bipolar cell pathway, we can make the following experimentally testable predictions.

First, brief movements of targets in the fish's environment will not be detected, whereas sustained moving targets will. The Pd stellate cell responses to electrosensory input will be subject to thresholding and conditional inhibition at the level of the ELL pyramidal cell.

Second, Iontophoresis of GABAB antagonists to the pyramidal cell/ventral molecular layer region will reduce the stimulus threshold for individual cells and interfere with normal signal from noise extraction. This could be tested by monitoring the feature vector extraction performance of individual cells (Gabbiani et al. 1996; Metzner et al. 1998).

Conditional inhibition needs to be included in models of ELL feedback for them to be biologically relevant. Intrinsic neuronal conductances (such as IA) are excluded from current network models of the electrosensory system (Payne et al. 1994), and they are therefore likely to severely underestimate the sophisticated computations that the electrosensory system is capable of making.

    ACKNOWLEDGEMENTS

  We thank W. Ellis for excellent technical assistance.

  L. Maler and N. J. Berman were supported by the Medical Research Council of Canada.

    FOOTNOTES

  Address for reprint requests: N. J. Berman, Dept. of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa ON K1H 8M5, Canada.

  Received 4 February 1998; accepted in final form 27 August 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society