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J Neurophysiol 80: 3173-3196, 1998;
0022-3077/98 $5.00
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The Journal of Neurophysiology Vol. 80 No. 6 December 1998, pp. 3173-3196
Copyright ©1998 by the American Physiological Society

Inhibition Evoked From Primary Afferents in the Electrosensory Lateral Line Lobe of the Weakly Electric Fish (Apteronotus leptorhynchus)

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. Inhibition evoked from primary afferents in the electrosensory lateral line lobe of the weakly electric fish (Apteronotus leptorhynchus). J. Neurophysiol. 80: 3173-3196, 1998. The responses of two types of projection neurons of the electrosensory lateral line lobe, basilar (BP) and nonbasilar (NBP) pyramidal cells, to stimulation of primary electrosensory afferents were determined in the weakly electric fish, Apteronotus leptorhynchus. Using dyes to identify cell type, the response of NBP cells to stimulation of primary afferents was inhibitory, whereas the response of BP cells was excitation followed by inhibition. gamma -Aminobutyric acid (GABA) applications produced biphasic (depolarization then hyperpolarization) responses in most cells. GABAA antagonists blocked the depolarizing effect of GABA and reduced the hyperpolarizing effect. The GABAB antagonists weakly antagonized the hyperpolarizing effect. The early depolarization had a larger increase in cell conductance than the late hyperpolarization. The conductance changes were voltage dependent, increasing with depolarization. In both cell types, baclofen produced a slow small hyperpolarization and reduced the inhibitory postsynaptic potentials (IPSPs) evoked by primary afferent stimulation. Tetanic stimulation of primary afferents at physiological rates (100-200 Hz) produced strongly summating compound IPSPs (~500-ms duration) in NBP cells, which were usually sensitive to GABAA but not GABAB antagonists; in some cells there remained a slow IPSP that was unaffected by GABAB antagonists. BP cells responded with excitatory or mixed excitatory + inhibitory responses. The inhibitory response had both a fast (~30 ms, GABAA) and long-lasting slow phase (~800 ms, mostly blocked by GABAA antagonists). In some cells there was a GABAA antagonist-insensitive slow IPSP (~500 ms) that was sensitive to GABAB antagonists. Application of glutamate ionotropic receptor antagonists blocked the inhibitory response of NBP cells to primary afferent stimulation and the excitatory response of BP cells but enhanced the BP cell slow IPSP; this remaining slow IPSP was reduced by GABAB antagonists. Unit recordings in the granule cell layer and computer simulations of pyramidal cell inhibition suggested that the duration of the slow GABAA inhibition reflects the prolonged firing of GABAergic granule cell interneurons to primary afferent input. Correlation of the results with known GABAergic circuitry in the electrosensory lobe suggests that the GABAergic type 2 granule cell input to both pyramidal cell types is via GABAA receptors. The properties of the GC2 GABAA input are well suited to their putative role in gain control, regulation of phasicness, and coincidence detection. The slow GABAB IPSP evoked in BP cells is likely due to ovoid cell input to their basal dendrites.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The importance of synaptic inhibition in processing sensory input is acknowledged, yet the functional role played by inhibition is still under debate (Berman et al. 1992; Martin 1988; Somers et al. 1995). The major inhibitory neurotransmitter, gamma -aminobutyric acid (GABA) has been shown to shape the responses of neurons in sensory systems (Sillito 1984), yet it has proved difficult to identify the inhibitory neurons and pathways responsible for the various response properties of sensory neurons. This is largely due to the complexity and number of inhibitory circuits present in the most commonly studied sensory structures, e.g., visual cortex. The electrosensory system of weakly electric fish is a far simpler sensory structure, which has been extensively studied from both a morphological (Carr and Maler 1986) and functional point of view (Bastian 1986a; Heiligenberg 1991). In the electrosensory lateral line lobe (ELL) of the weakly electric fish, we have identified GABAergic neurons and pathways that are likely to be involved in electroreception (Maler and Mugnaini 1994). We anticipate that attempts to correlate inhibitory pathways with particular sensory processing functions will be more successful in this system and will help to focus investigations in other sensory systems.

The gymnotiform species, Apteronotus leptorhynchus, has a neurogenic electric organ in its tail that generates a high-frequency electric organ discharge (EOD) (reviewed by Bass 1986). The EOD of this species resembles a sine wave ranging from 500 to 1,200 Hz (at 28°C) and is constant except when used intermittently for communication (Bullock 1969; Zupanc and Maler 1997). The fish has specialized electroreceptors distributed on its body surface that detect changes in the electric field surrounding the fish. There are two types of electroreceptors (see Carr and Maler 1986; Zakon 1986 for review): ampullary receptors (~360) (Carr et al. 1982) detect slowly varying exogenous electric fields, whereas tuberous receptors (~6,500-8,500) (Carr et al. 1982) are tuned to the EOD of the individual fish. Tuberous receptors are divided into two types: phase coders (T-units) fire 1:1 with each cycle of the fish's EOD, whereas probability coders (P-units) scale their firing rate according to the local amplitude of the EOD. A. leptorhynchus has few phase coders, and so these are not discussed here (see Mathieson et al. 1987). Probability coders are essential for both electrolocation and electrocommunication (Bastian 1986a; Heiligenberg 1991).

The sole recipient of electroreceptor afferents is the ELL (see atlas of Maler et al. 1991 for location). The ELL consists of four independent segments, each containing a topographic electrosensory map (Carr et al. 1982; Heiligenberg and Dye 1982). One segment (medial segment: MS) receives only ampullary input, whereas the other segments (centromedial: CMS, centrolateral: CLS, lateral: LS) receive tuberous input (mostly P units). There are physiological and morphological differences among the segments (Metzner and Juranek 1997; Shumway 1989a,b; Turner et al. 1996) but all three tuberous segments contain similar canonical circuits of excitatory and inhibitory neurons and pathways (Fig. 1) (Fig. 2 in Maler and Mugnaini 1994). This and the companion studies (Berman and Maler 1998a,b) were conducted exclusively in the CMS; hence all results are interpreted for this segment only, as intrinsic cell properties and proportions of cell types vary across the segments. All four segments of the ELL have a similar laminar structure consisting of: a deep fiber layer (DFL) consisting mainly of electrosensory afferents, a granular cell layer (GCL) of interneurons, a pyramidal cell layer (PCL), and dorsal and ventral molecular layers (DML, VML) containing apical dendrites of pyramidal cells, interneurons, and feedback input from higher brain centers. The electroreceptor afferents terminate in a deep neuropil region (ventral to the granular cell layer) where they form chemical synaptic contacts with the basal bush of basilar pyramidal neurons, and mixed (chemical and gap junction) synapses onto dendrites of several types of putative inhibitory interneurons: type 1 granular cells (GC1; transmitter not identified), type 2 granular cells (GC2; GABAergic), ovoid cells (GABAergic) and polymorphic cells (GABAergic) (Maler 1979; Maler and Mugnaini 1994; Maler et al. 1981; Yamamoto et al. 1989).


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  		FIG. 1. Topography and relevant inhibitory circuitry of the electrosensory lateral line lobe (ELL). A: coronal section (cresyl violet stain) from the atlas of Maler et al. (1991; level T-7). ELL layers are approximately indicated for the segment investigated in this study: the centromedial segment. CCb, cerebellum; mol, molecular layer; StF, stratum fibrosum; PCL, pyramidal cell layer; GCL, granule cell layer; DNL, deep neuropil layer; DFL, deep fiber layer. B: circuit diagram of pyramidal cell types and the relevant inputs that could be driven by stimulation of the DFL. Nonbasilar and basilar cells: both receive GABAergic input from type 2 granule cells (GC2) (Maler and Mugnaini 1994) and inhibitory (based on synapse ultrastructure) (Maler et al. 1981) input of unknown transmitter type from type 1 granule cells (GC1). Basilar pyramidal cells differ only in that their basilar dendrites receive GABAergic input along its length from ovoid cells and direct excitatory (glutamatergic) input from the primary afferents.


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  		FIG. 2. Morphology and physiology of pyramidal cells. A: montage of fluorescent confocal images of a basilar (BP, left) and nonbasilar pyramidal cell (NBP, right) recorded in different transverse ELL slices. Approximate lamina boundaries for both slices are indicated (- - -). GCL, granule cell layer; pl, plexiform layer; VML, ventral molecular layer; DML, dorsal molecular layer. Labels near the somas (a and b) and the basilar dendritic bush (c) indicate the regions scanned at high magnification in Fig. 3. B: intracellular responses (average of 3 sweeps) at rest (BP: -68 mV; NBP: -66 mV) of each cell in A to a single stimulus (20 us, 70 µA) applied to the DNL ~300 µm ventral to the granule cell layer. Stimulus artifacts (black-triangle) have been removed in this and all subsequent figures for clarity. C: intracellular responses of same cells to tetanic stimulation (200 Hz, triangles) at rest (bottom) and during current injection (+0.2 nA, top). Both cells were quiescent at rest. Responses of BP cell are to single trials; NBP traces are averages of 3 trials. Spike heights truncated by cropping and/or averaging in this and subsequent figures.

The principal output neurons of the ELL are the pyramidal cells (Fig. 1). There are two morphologically and physiologically distinct types of ELL pyramidal neurons: basilar (BP) and nonbasilar pyramidal cells (NBP). BP cells (E units) receive direct glutamatergic synaptic input from tuberous afferents onto their basal dendrite and are excited by increases in EOD amplitude (Maler 1979; Maler et al. 1981; Saunders and Bastian 1984; Wang and Maler 1994); this cell type also receives an input from the GABAergic ovoid cell, which climbs on its basal dendrite as well as input from GC1 and GC2 cells onto its soma (Maler and Mugnaini 1994). NBP (I units) somata and somatic dendrites receive input from GABAergic and non-GABAergic granular cells (GC2 and GC1) and are excited by decreases in EOD amplitude (Maler 1979; Maler and Mugnaini 1994; Maler et al. 1981; Saunders and Bastian 1984). The descending excitatory and inhibitory inputs to pyramidal cells, which terminate in the dorsal laminae of the ELL (Carr and Maler 1986; Maler and Mugnaini 1993, 1994) are discussed in the accompanying papers (Berman and Maler 1998a,b).

The responses of pyramidal cells to sensory input bear remarkable resemblance to those of mammalian sensory systems, such as the retina. ELL pyramidal cells have receptive fields that have center-surround antagonism (Shumway 1989a) and flexible gain control/contrast adaptation (Bastian 1986b, 1995, 1996). Maler and Mugnaini (1994) have attempted to relate ELL inhibitory circuitry to its sensory physiology. The inhibitory input to pyramidal cells from GABAergic GC2 was proposed to control the size and strength of receptive field centers (Maler and Mugnaini 1994). They further hypothesized that non-GABAergic input from GC1 contributes to surround regulation. Ovoid cell input to BP cells is thought to mediate common mode rejection (see Bastian et al. 1993; Maler and Mugnaini 1994). However, little is known of the mechanisms and dynamics of inhibition in the ELL. Such data are needed to assess the validity of these hypotheses. In this paper we have investigated the mechanisms of inhibition that may underlie the first stage of electrosensory processing in the ELL. This study identifies the biophysical and pharmacological nature of inhibitory responses recorded in pyramidal cells in response to stimulation of the DFL. Our results show that BP and NBP cells can be identified by their response to DFL stimulation and that both GABAA and GABAB receptors are involved in their inhibitory responses. GC2 are likely to generate mostly GABAA-receptor-mediated inhibition, whereas ovoid cell input is mediated partly by GABAB input to BP cells.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Slice preparation

Slices of ELL were prepared according to a modified method of Mathieson and Maler (1988) and Turner et al. (1994). Briefly, fish of either sex were anesthetized by immersion in water containing 0.2% 3-aminobenzoic acid ethyl ester (MS-222; Sigma provided all chemicals unless otherwise indicated). All surgical procedures were approved by the University of Ottawa Animal Care Committee. The fish then were transferred to a foam-lined clamp, and a glass tube was introduced into their mouth that supplied aerated water containing the same anesthetic. The brain was exposed by dissection of the skin and overlying skull while the surgery site was irrigated continuously with chilled artificial cerebrospinal fluid [ACSF, which contained (in mM) 124 NaCl, 3 KCl, 0.75 KH2PO4, 2 CaCl, 2 MgSO4, 24 NaHCO3, and 10 D-glucose]. Care was taken to avoid touching the ELL while underlying nerves were cut. The brain was blocked to ensure a true transverse section of the ELL by cutting the rostral face of the block at ~45° angle at the level of the optic tectum. The brain block was removed from the skull after severing the spinal cord, glued to a chilled aluminum chuck with cyanoacrylate adhesive, and covered with warm (~25°C) low gelling temperature agarose (5% in ACSF; FMC, Rockland, ME). The chuck was placed immediately in a Vibratome (modified to provide high-amplitude blade vibrations, Technical Products International, St. Louis, MO), immersed in chilled carbogenated (95% O2-5% CO2) ACSF and cut into 350-µm sections. As each section was cut, it was sucked up into a wide-mouthed plastic pipette and ejected into a holding chamber containing carbogenated and chilled ACSF. Slices then were transferred to an interface type slice chamber where they were maintained at room temperature (21-23°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. Recordings were started after a 1- to 2-h recovery period.

Stimulation and recording

Electrical stimulation of the primary afferents of the ELL was achieved by placing a lacquer-coated sharpened tungsten electrode (50 µm exposed tip) into the DFL region of the CMS, lateral to the recording site in the PCL (see Fig. 1). Square wave pulses (50-100 µs, 1-50 V or 20-600 µA cathodal, NL102 or SIU unit, Digitimer, UK) were delivered via the stimulating electrode. Stimulus intensity was adjusted to evoke ~70% of maximal response amplitude. Excessively high stimulation parameters caused direct or antidromic activation of the cells. Intracellular recordings were made in the CMS PCL with 2 M potassium acetate-filled electrodes bevelled to 55-120 MOmega DC resistance. For recovery of cells, 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 and bevelled to 120 MOmega ) pipettes. All experiments were software controlled (PCLAMP, Axon Instruments, Foster City, CA or A/Dvance, Rob Douglas, University of British Columbia, Vancouver BC, Canada). Electrical activity was amplified (Axoclamp, Axon Instruments), filtered (10 kHz fc), digitized (1.25-50 kHz) and analyzed off-line (Igor Pro 3.0, Wavemetrics, OR; Macintosh PowerPC, Apple, CA).

Drug applications

For iontophoretic applications, drugs were dissolved in 100 mM NaCl at the following concentrations: GABA and baclofen (GABAB agonist) 0.1 M [Research Biochemicals International (RBI), MA], 5 mM bicuculline (GABAA antagonist: Tocris Cookson, UK; RBI, MA; Sigma, MO). For application to the slice by microdroplet, antagonists were dissolved in ACSF at the following concentrations: 70 µM bicuculline, (Tocris Cookson; RBI; Sigma), 100 µM SR(-95531) (GABAA antagonist: RBI), 0.05-1 mM [6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor antagonist: RBI], 2 mM DL-2-amino-5-phosphonovaleric acid (APV) [N-methyl-D-aspartate (NMDA)-receptor antagonist 1 CPP (±)-3-2(2-carboxypiperazin-4-gl)-propyl-1-phosphonic acid, NMDA antagonist, RBI, MA; Tocris Cookson], and 1-3 mM phaclofen and saclofen (GABAB antagonists: RBI; Tocris Cookson]. Microdroplets were applied by pressure via a broken-back pipette (10-20 µm) so that a single droplet covered an area of the slice surface ~200-400 µm in diameter. This ensured that most of the input to the recorded cell, from the GCL to the PCL, would be exposed to the drug. Iontophoretic pipettes were inserted into the PCL as close as possible to the estimated recording site both in lateral distance and recording depth. Depth was adjusted to gain the best response to a test GABA pulse.

Histology and reconstruction

After recording, slices were allowed to incubate for >= 1 h before fixation. Slices, which lay on individual pieces of lens tissue paper (Kodak, Rochester, NY), were removed from the chamber, placed on a glass slide, and had a droplet of fixative applied [4% paraformaldehyde 0.5% glutaraldehyde in 0.1 M phosphate buffered saline (PBS)]. Slices were postfixed at room temperature for 1-3 h and then stored in PBS overnight. The slices were resectioned on a vibratome at 80 µm, placed on glass slides, dried, and processed according to a modified Horikawa and Armstrong (1988) protocol. Background peroxidase activity was removed by pretreatment of slides with 0.3% H2O2 in PBS. Horseradish peroxidase was revealed by using a cobalt-nickel-intensified (Adams 1981) diaminobenzidine (0.2 mg/ml in 0.1 M sodium acetate buffer, pH 6.0) procedure. Treatment with 0.1% (in PBS, 30-90 s) Osmium tetroxide further darkened the reaction product. Sections then were dried, defatted, and cover-slipped. For Lucifer yellow visualization, slices were fixed with paraformaldehyde (4% in PBS). Sections were cleared using dimethyl sulfoxide to minimize shrinkage distortions (Grace and Llinas 1985). Sections were viewed on a fluorescence microscope (BH-2; Olympus America, NY). Pyramidal cells were identified as basilar versus nonbasilar on the basis of the presence of a basal dendrite (Maler 1979). For illustration, sections were scanned on a confocal microscope in 5- or 10-µm optical slices (×16 averaging). Final montages were made by merging all optical slices (Photoshop Version 5.0, Adobe Systems, CA) using the "lighten" algorithm (each pixel has the highest value sampled from each optical slice).

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Biophysical parameters

Intracellular recordings were obtained from cells impaled in the PCL of ELL slices. This study concentrates on the inhibitory responses that are evoked by DFL stimulation; this inhibition must arise from activation of three inhibitory interneurons: GC1, GC2, and ovoid cells (Maler and Mugnaini 1994) are the only known interneurons that receive direct primary afferent input via glutamatergic and gap junction synapses (Maler 1979; Maler et al. 1981; Mathieson et al. 1987) and project to pyramidal cells.

Of the 101 cells impaled in the PCL, 15 cells were histologically identified as BP (n = 9) or NBP (n = 6). Based on their electrophysiological response to DFL stimulation (see next section), a further 78 cells were classified as either BP (n = 49) or NBP (n = 29). The remaining cells could not be assigned unambiguously to either group or were suspected to be polymorphic type cells (see following text) and were not included in the sample. The summary statistics of the pyramidal cells' passive and active biophysical characteristics are presented in Table 1. There were no significant differences between the means of the BP and NBP cell groups. The passive parameters are unremarkable and are similar to those reported for ELL pyramidal cells (Berman et al. 1997; Mathieson and Maler 1988; Turner et al. 1994) and for pyramidal cells in the benchmark rat hippocampal slice preparation (Schwartzkroin 1975).

 
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  		TABLE 1. Summary of biophysical parameters of cells recorded in the ELL slice preparation

ELL BP and NBP spikes had a half-width (0.4 ms) narrower than typical cortical or hippocampal pyramidal cells (~0.5-0.6 ms) (Mason and Larkman 1990). Two cells, not included in Table 1, had very narrow spikes (half-width <0.18 ms), a large afterhyperpolarization (AHP; ~10 mV), and rapid accommodation of spike rate to injected current. These spike characteristics have been observed in identified polymorphic cells (Maler 1979; R. Turner, personal communication). Twelve cells probably were impaled in their dendrites because their current-evoked spikes decayed slowly [1.00 ± 0.47 (SE) ms], were wide (half-width 0.617 ± 0.147 ms), and lacked a fast AHP (see Turner et al. 1994).

Basilar and nonbasilar pyramidal cells can be identified by their response to DFL stimulation

MORPHOLOGY. Histological recovery of Lucifer yellow (LY)- or biocytin-filled cells were used to correlate their responses to DFL stimulation with their classification as BP or NBP cells. The cellular morphology and intracellular responses of a typical LY-filled BP cell is shown in Figs. 2 and 3. The soma of the BP cell straddled the pyramidal and plexiform layers. In this case the sparse somatic dendrites arose from the apical dendrite trunk within the pyramidal cell layer (the site of termination of granular cell axons) (Maler 1979; Maler and Mugnaini 1994). The axon can be seen leaving the soma and coursing horizontally through the plexiform layer (Fig. 2A). The thick apical dendrite courses through the stratum fibrosum and ramifies in the molecular layer. The thick basal dendrite courses ventrally through the plexiform and granular cell layers to form a compact but dense bush in the deep neuropil layer (Fig. 3C), where it receives excitatory input from primary afferents (Maler et al. 1981). Our fills demonstrated that the basal bush was far denser than seen in typical golgi impregnations (Maler 1979).


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  		FIG. 3. High magnification views of regions of BP and NBP cells indicated in Fig. 2. A: BP cell soma. right-arrow, site of axon hillock and axon emerging from the soma. Axon can be followed for several hundred micrometers in Fig. 2A (left). There is a plexus of fine (0.2-2 µm diam) varicose dendrites that arise from the most dorsal region of the soma and the proximal apical dendrites. These are confined to the PCL. B: NBP cell soma. right-arrow, axon emerging from ventral tip of soma (see Fig. 2A, right). Axon continue in the plexiform layer. NBP cell soma has a denser but otherwise similar plexus of fine varicose dendrites. In this case, these fine dendrites arise from the soma only. C: basilar dendrite bush. Basilar dendrite terminates in a dense varicose plexus with process diameters ranging from 8 to 0.2 µm.

The NBP cell soma was situated within the pyramidal cell layer (Fig. 2A) and had a more extensive somatic dendritic arbor than the BP cell (cf. Fig. 3, A with B); this was typical of our sample and corresponds with our previous Golgi description of these cells [although the previous studies (Maler 1979; Saunders and Bastian 1984) did not allow full resolution of these fine processes]. Because these processes receive rich GABAergic input (Maler and Mugnaini 1994; Maler et al. 1981), this implies that NBP cells might have more potent inhibitory responses than BPs. The functional implications of placement of GABAergic inputs on these extensive and fine somatic arbors were further explored by computer simulation (see APPENDIX ). The apical dendritic morphology of NBP and BP cells was indistinguishable (Maler 1979; Saunders and Bastian 1984).

PHYSIOLOGY. We recorded the intracellular responses of the same cells to single (Fig. 2B) and tetanic (200 Hz; Fig. 2C) stimulation of the primary afferents via an electrode in the DFL. This BP cell responded to a single stimulus with a short-lasting excitatory postsynaptic potential (EPSP) (<10 ms half-width, Fig. 2B, left) followed by a longer lasting IPSP; when it appeared, the IPSP was always associated with a preceding EPSP. The NBP cell responded with an IPSP alone (2 ms onset, 14 ms to peak, Fig. 2B, right). Postsynaptic potentials were classified as EPSPs if they were depolarizing at resting membrane potential (Vm) and, when the cell was depolarized with current injection, increased the probability of spike firing. The polarity of IPSPs depended on resting Vm; cells with large (approximately less than -75 mV) resting Vm had depolarizing IPSPs that reversed with depolarizing current injection and reduced the probability of spike firing (see following text).

Tetanic stimulation (Fig. 2C) produced a summating series of EPSPs or compound EPSP in the BP cell and a summating series of IPSPs or compound IPSP in the NBP cell. The cells in Fig. 2 were tested both at rest and during injection of current (+0.2 nA).

The BP cell did not fire at rest, and tetanic stimulation (Fig. 2C, bottom) could evoke spikes only on the last few EPSPs in the tetanus; tetanic stimulation was effective in producing six spikes in the depolarized condition (Fig. 2C, left, top). Higher stimulation intensities were capable of synaptically evoking spikes at rest both to single stimuli and to every stimulus in the tetanus (not shown). The depolarized condition (Fig. 2C, left, top) revealed a slow IPSP following the compound EPSP. Depolarizing current injection (<= 0.6 nA) induced discharge in this cell that was blocked by the slow IPSP. The NBP also fired during current injection, and this discharge was completely suppressed both during the tetanus and during the evoked slow IPSP (>300 ms).

The preceding results were representative for nine BP and six NBP cells filled with either HRP or LY. BP cells always displayed a clear short-latency EPSP (0.96 ± 0.48 ms, n = 8) or compound EPSP to tetanic stimulation (3 of 3 cells tested), whereas filled NBP cells displayed a short-latency IPSP (2.8 ± 0.4 ms to peak, n = 4) or compound IPSP to tetanic stimulation (3 of 3 cells tested). In one filled NBP cell, we observed a short-latency depolarizing potential (0.4 ms to peak) that was presumably due to gap junction input from granular cells (Maler et al. 1981). The shape and amplitude of this potential remained constant at all membrane polarizations, unlike the EPSPs in BP cells the amplitudes of which were voltage sensitive (not shown).

Pyramidal cell response to exogenous GABA and baclofen

RESPONSE TO GABA. GABA was ionophoresed (1-10 s, 10-200 nA) within the PCL close to the intracellular recording site for 13 physiologically identified pyramidal cells while membrane potential and input resistance (Rin) changes were monitored. Both BP and NBPs displayed similar responses to GABA at the resting Vm (Fig. 4); a rapid initial hyper- or depolarization followed by a lower amplitude sustained hyperpolarization. The initial response was brief (0.5-4 s), and its polarity dependent on resting Vm (Figs. 4 and 5). It was depolarizing in 8 of the 13 cells.


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  		FIG. 4. Response of BP cell to gamma -aminobutyric acid (GABA) application. A1: response to GABA at 3 membrane potentials (resting Vm = -61 mV). Input resistance (Rin; control = 22 MOmega ) was monitored by injecting -0.5-nA current pulses (50 ms, 1 Hz) and measuring the resulting voltage deflections. - - -, control membrane potential. DC currents were injected into the cell to depolarize (top) and hyperpolarize (bottom) the cell from rest (middle). This and subsequent figures: short depolarizing voltage deflections were caused by 0.5-nA pulses used to monitor cell excitability (firing response). These deflections have been truncated digitally. A2: normalized Rin measurements derived from responses to hyperpolarizing current pulses in A1. B1: same data as in A, but with voltage deflections digitally removed and all 3 traces plotted on same voltage axis. This plot indicates >= 2 components to the GABA response. Middle: hyperpolarizing peak at 1 s after the onset of GABA. Bottom: depolarizing peak at 5 s. Markers indicate latency from onset of GABA application where measurements (plotted in B2) of the GABA response were made. B2: measurements of the GABA response magnitude vs. the control holding potential indicate the approximate reversal potential (membrane potential where the interpolated GABA response would be 0 mV) of the early (square ; -65 mV) and late (bullet ; -62.5 mV) components of the GABA response.


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  		FIG. 5. Effect of GABAA antagonist on the response of a pyramidal cell to GABA. A1: response of pyramidal cell to 5 s (middle: rest Vm; top: depolarized) and 10 s (bottom: hyperpolarized) GABA iontophoresis (50 nA). Rin and excitability monitored as in Fig. 5. Control membrane potential is indicated (- - -). A2: absolute Rin change during the GABA responses at the 3 polarization levels in A1. right-arrow, 2 peaks (2 and 6 s) in the time course of the decrease of Rin. B1: response of the same cell to GABA applications after microdroplet application of 50 µM SR. Early depolarizing component of the response is blocked, whereas the slower hyperpolarizing component is unaffected by SR. B2: changes in Rin during the responses in B1. First peak arrowed in A2 is reduced by SR. Extrapolated reversal potentials: GABA depolarizing response in A1 = -40 mV, GABA hyperpolarizing response (measured at GABA offset) was -94 mV in before and -100 mV during the SR treatment.

Polarizing the cell with injected current revealed that this initial response may be composed of more than one phase with reversal potentials near rest (Fig. 4B). The small hyperpolarization seen at resting potential reversed at -65 mV. A second phase appeared as a later depolarizing peak (Fig. 4B; hyperpolarized trace, bullet ) with a slightly more depolarized reversal potential (-62.5 mV). The peak of the second component coincided with the greatest decrease in Rin (Fig. A2). After the iontophoretic current was turned off, a small hyperpolarization (0.5-1 mV) persisted for <= 15 s. The slow response, which outlasted the GABA application, was of longer duration (>4 s) and did not reverse polarity even in the most hyperpolarized traces (<-86 mV; Fig. 5).


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  		FIG. A2. Temporal characteristics of somatic dendrite inhibition. A: single IPSP activations (t = 0 ms, Alpha function time to peak = 2 ms). Active compartments indicated (see Fig. 1.). Top: soma voltage plots; bottom: soma and somatic dendrite (trunk only) synaptic conductances. Note that the IPSP voltage change tracks closely the conductance change. There is little evidence of the IPSP voltage change outlasting the conductance change (somatic dendrite trunks = 2.5 µm diam). Steady depolarizing current, 0.5 nA. B: tetanic IPSP activation (10 pulses, 200 Hz). Tetanus activation produced a summating compound IPSP. Smoothness depended on the time to peak of the IPSP alpha function (2 and 5 ms tested). Various traces are from simulation runs in which the somatic dendrite tips alone or with somatic dendrite trunks or all synaptic conductances were activated (see Fig. A1, A).

GABA produced a large decrease in Rin throughout the applications irrespective of whether the initial response was hyper- or depolarizing (Figs. 4-6); the postapplication hyperpolarization was accompanied by a much smaller decrease in Rin (Figs. 4 and 5). The conductance change underlying the GABA response was strongly voltage dependent (e.g., Fig. 4. A2); in this experiment, Rin decreased by <= 2.5 MOmega when the cell was hyperpolarized and GABA applied, whereas when the cell was depolarized to -55 mV, Rin decreased by as much as 7 MOmega during the GABA application. Although all pyramidal cells showed some voltage dependency (rectification) of their steady-state Rin, this could not account for the voltage dependence of the GABA response. Indeed, the hyperpolarizing response to GABA (most depolarized case) would offset the intrinsic voltage dependency of Rin, therefore voltage dependence of the GABA response may be underestimated. The cell in Fig. 4 was typical in that control Rin decreased with depolarization and vice versa (compare control period voltage deflections at the 3 membrane potential levels). This rectification was probably due to the voltage dependence of intrinsic currents (e.g., IA and INap) (see Mathieson and Maler 1988) and is not discussed further. The change in Rin produced by GABA scaled with iontophoretic current, ejection time, and proximity of the drug electrode to the impalement site; Rin became unmeasurably low with large GABA ejection parameters.


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  		FIG. 6. Response of pyramidal cell to GABA and baclofen in the presence of SR314 and saclofen. A: GABA (76 nA, 10 s) applied during control conditions produced a strong hyperpolarization of the cell. After a microdroplet application of saclofen (2 mM), the response to GABA was diminished (middle). When SR314 also was applied to the pyramidal cell layer, the GABA response was blocked almost completely. Note the presence of spontaneous slow membrane potential oscillations during control and saclofen conditions. SR314 enhanced the oscillations causing the cell to fire action potentials on the depolarizing phase. Action potentials are truncated in the figure. B: Rin measurements derived from the data in A. Saclofen reduced the Rin change during GABA applications. Under saclofen and SR314 conditions GABA did not change Rin appreciably. Oscillations caused fluctuations in control Rin that were accentuated during the SR314-induced large amplitude oscillations. Saclofen alone had no effect on the oscillations. C: under control conditions, 20-s iontophoresis (150 nA) of baclofen caused a slow hyperpolarization from rest (- - -). Prior application of saclofen did not antagonize the baclofen response (bottom). D: change in Rin during the baclofen responses in A. Baclofen caused a small decrease in Rin (~0.5 MOmega ) that, if anything, was enhanced by the presence of saclofen.

EFFECT OF GABAA ANTAGONISTS ON THE GABA RESPONSE. To determine whether the responses to GABA were due to activation of GABAA receptors, bicuculline (70 µM) or SR (50 µM) were applied by microdroplet to the pyramidal cell layer prior to iontophoresis of GABA (Fig. 5). The efficacy of the antagonists was apparent after several seconds by the appearance of membrane potential oscillations (Turner et al. 1991). Both GABAA antagonists blocked or substantially decreased the initial (hyper- or depolarizing) part of the GABA response (n = 8). In the example in Fig. 5, the GABA response was initially depolarizing followed by a longer lasting hyperpolarization. Note that the cell's resting potential was -74 mV and was not polarized (with injected current) beyond the GABA depolarizing response's reversal potential (-56 mV by extrapolation). SR blocked the depolarizing response but did not affect the longer hyperpolarization (Fig. 5B1) the extrapolated reversal potential of which was -100 mV. This hyperpolarization, which under SR block started at the onset of the GABA response, probably was masked by the depolarization under control conditions. The Rin changes underlying the GABA responses (Fig. 5, A2 and B2) showed typical strong voltage dependency. The SR block of the depolarizing response was associated with a block of the early peak conductance shift seen in control conditions (1st right-arrow in Fig. 5, A2 and B2), while the second peak in the conductance shift was relatively unaffected by SR (2nd right-arrow, Fig. 5, A2 and B2).

GABAB-MEDIATED RESPONSES TO GABA AND BACLOFEN. Because the slow GABA-evoked hyperpolarization was insensitive to bicuculline or SR, GABAB antagonists (saclofen or phaclofen) and an agonist (baclofen) were tested on the pyramidal cells. Figure 6 shows one such experiment; in this cell (which displayed a small oscillation of membrane potential under control conditions) both components of the GABA response were hyperpolarizing at rest. Treatment with saclofen (2 mM) curtailed mainly the duration of the hyperpolarization. Conductance measurements showed a decrease in the magnitude of the GABA-induced conductance increase (Fig. 6B, ···). Treatment with both SR and saclofen before the GABA application blocked both the voltage and conductance shifts. Thus at least part of the GABA response was sensitive to the GABAB antagonist. GABAB antagonists clearly affected part of the GABA response in only 5 of 12 cases.

The GABAB agonist baclofen consistently (n = 13) produced a small (1-2 mV) hyperpolarization and increase in spike current threshold (not shown). The conductance change underlying the response was either small (<= 1 MOmega ) or undetectable. This is expected because the effect of rectification with hyperpolarization (which would tend to increase Rin) would cancel the small conductance increase associated with the response to baclofen. Treatment of the slice with microdroplets of saclofen (n = 3, 2 mM) was unable to antagonize the response to baclofen. In the example shown in Fig. 6, the cell depolarized slightly (by 4 mV) after saclofen treatment; this actually increased the magnitude of the response to baclofen.

These experiments demonstrated that both types of pyramidal cells respond with both GABAA and GABAB-like potential and conductance changes and pharmacology, as seen in mammalian pyramidal cells in vitro (hippocampus: Alger and Nicholl 1979, 1982; Scharfman and Sarvey 1988b; neocortex: Scharfman and Sarvey 1988a). Thus although a molecular characterization is presently lacking, we conclude that ELL pyramidal cells (BP and NBP) have both GABAA and GABAB receptors; traditional GABAA antagonists are highly effective in the ELL, whereas the GABAB antagonists (saclofen and phaclofen) appear to have a relatively low potency.

EFFECT OF BACLOFEN ON INHIBITION. GABAB receptors have been shown to decrease presynaptic release of GABA in other systems (Thompson and Gähwiler 1992). This was tested in the ELL by observing the effect of baclofen treatment on the inhibition evoked by DFL stimulation (NBP cells, n = 3). Under control conditions, tetanic stimulation produced strong hyperpolarizing inhibition (Fig. 7A, right-arrow). The cell was current-clamped at various potentials to monitor the efficacy of the inhibition. After baclofen treatment, DFL stimulation did not evoke strong inhibition. To test whether the baclofen-induced postsynaptic decrease in Rin could account for the disappearance of the stimulus-evoked inhibition, the cell was depolarized to levels that previously had produced strong repetitive firing (Fig. 7, B and C). Under control conditions, the stimulus-evoked inhibition potently reduced the firing rate of the cell (Fig. 7C, right-arrow), however, after baclofen treatment, it had only a small inhibitory effect on spike rate (Fig. 7, B and C, right). We conclude that GABAB receptors are likely to be present on GABAergic nerve terminals in the PCL.


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  		FIG. 7. Effect of baclofen on tetanic stimulus evoked inhibition of a nonbasilar pyramidal cell. Left: data collected under control conditions. Right: data collected after microdroplet of baclofen (50 µM). A: response of the NBP cell to tetanic stimulation (100 Hz, shaded area) of the DFL during current clamp (-0.75-0.25 nA, 720 ms) above and below resting Vm (-62 mV). Under control conditions (left), the depolarizing current evoked 1 spike before the tetanic stimulation produced a large inhibitory hyperpolarization (right-arrow). After baclofen application, the tetanic stimulation failed to evoke the strong inhibition. Some inhibition persisted as evidenced by the pause in firing after cessation of the tetanic stimulus (top). B: response to the same tetanic stimulation during injection of strong depolarizing current (0.7 nA) that evoked repetitive firing of action potentials. Effect of the tetanic stimulus on the firing rate is plotted in C. C: firing rate derived from data in B. Under control condition, right-arrow, drop in firing rate coinciding with the peak inhibition seen in A. Firing rate during the baclofen condition (right) is compared with the control response replotted (- - -).

Electrophysiological and pharmacological characteristics of synaptically evoked inhibition in NBP cells

ELECTROPHYSIOLOGY OF NBP CELL INHIBITION. As shown in Fig. 2, the response of NBP cells to DFL stimulation was inhibitory. IPSPs could be classified into at least two categories, fast and slow. Fast IPSPs peaked at between 1.8 and 25 ms (mean 8.4 ± 8.7 to peak, n = 28), whereas slow IPSPs peaked at between 150 and 600 ms (mean 501.3 ± 371.7 ms to peak, n = 20) and endured for <= 2 s. The slow IPSP was absent in 26% of NBP cells. The fast IPSP reversed at -71.1 ± 7.1 mV (n = 8); this is more negative than the reversal potential of the early phase of the response to GABA application (-65 mV; recall that the early phase of the response to exogenous GABA lasts >500 ms and may be a combination of >= 2 components). The slow IPSP reversed at -83.9 ± 6.5 mV, which is very negative to the GABAA (antagonist sensitive) hyperpolarization caused by exogenous GABA. The probably mixed origin of the slow IPSP is discussed later (see GABAa antagonism).

In vivo, the probability coding receptors (P units) of high-frequency wave species have high spontaneous firing rates (~200-400 spikes/s) and stimulus-evoked firing rate increases of several hundred spikes per second (Bastian 1981a; Xu et al.1996). Although single stimulus pulses could evoke IPSPs in the slice preparation, more physiological tetanic stimulation (100-200 Hz) was far more reliable in evoking both fast and slow IPSPs. An example of a NBP cell that responded well to both single and tetanic stimulation is shown in Fig. 8. Single-pulse stimulation at rest evoked a hyperpolarization that appeared to consist of at least two components, a fast and a slow IPSP (Fig. 8, A and C). The fast IPSP was strongly inhibitory; it prevented spike firing during depolarization, albeit for only a few milliseconds. The slow IPSP was only weakly inhibitory. Tetanic stimulation, however, produced a summating IPSP that was strongly inhibitory; it prevented firing in the face of strong depolarizing currents (<= 0.6 nA).


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  		FIG. 8. NBP cell response to single-pulse and tetanic PA stimulation during current injection. A: response to single stimulus pulse. At rest (-62 mV), a single stimulus evokes a fast (peak at 3 ms) and slow inhibitory postsynaptic potential (IPSP; peak at 120 ms). Current injection (0.15-nA increments) reversed the fast IPSP at about -66 mV. Top 2 traces: (in this and subsequent figures, traces with spikes will be displaced vertically and spikes truncated for clarity) only the fast IPSP has an appreciable effect on spike firing probability. B: response of same cell to tetanic (200 Hz, black-triangle and shaded area) stimulation. Summating IPSP during the tetanus and the slow IPSP after stimulation flatten but do not fully reverse during the hyperpolarizing current injection. Top: evoked summed IPSP is strongly inhibitory; spikes cannot be evoked during the summed IPSP. Note the voltage-activated conductances on the depolarized traces (see Mathieson and Maler 1988). C: expanded time-base trial shows 2 distinct phases to the IPSP(s) evoked by single-pulse stimulation.

The slow IPSPs were variable in duration and effect. Figure 9 shows a NBP that was spontaneously active. Single-pulse stimulation (Fig. 9A) evoked a slow IPSP that was moderately inhibitory (some spikes "broke" through after a few hundred milliseconds). Tetanic stimulation (Fig. 9B) evoked a large, strong summating IPSP that prevented spiking for <= 1.5 s. Note the tight clamping of the membrane potential during the early part of the summating IPSP.


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  		FIG. 9. Response of NBP cell to single and tetanic stimulation. A: responses to single stimulus (5 trials superimposed). Cell exhibited spontaneous voltage oscillations. Stimulus triggered a slow IPSP (peak at ~120 ms), and spikes could "break through" the inhibition after ~300 ms. B: response of same cell to tetanic stimulation (200 Hz; 7 trials superimposed). Stimulus evokes a large, rapidly summating IPSP that takes >1.3 s to fully recover. Note that the cell is inhibited for longer than in A; spikes do not occur during the decaying IPSP.

PHARMACOLOGY OF NBP CELL INHIBITION. NBP cells receive inhibitory input from at least two cell populations, GC1 and GC2, of which the latter are GABAergic (Maler and Mugnaini 1994). GABAA (bicuculline, picrotoxin, or SR) and GABAB (phaclofen and saclofen) antagonists were applied to the slice to identify the receptor subtypes responsible for the IPSPs evoked by DFL stimulation.

GABAA ANTAGONISM. In all cases, applications of the GABAA antagonists either induced slow oscillations (Turner et al. 1991) or increased the oscillation amplitude of spontaneously oscillating cells. The oscillation cycles consist of a slowly depolarizing ramp, which leads to spike firing; this then is terminated by a rapid hyperpolarization followed by the depolarizing ramp of the next cycle (Fig. 6A) (see Turner et al. 1996 for details). As GABAA antagonists did not block the rapid hyperpolarization of the oscillation cycle, it is unlikely to be due to GABAA receptors.

Both bicuculline (n = 7) and picrotoxin (n = 1) blocked the fast IPSPs evoked by single stimuli or tetani. The slow IPSP was reduced in four of the eight cells by GABAA antagonists (e.g., Fig. 10, A and C), and the remaining hyperpolarization was unaffected by the GABAB antagonist saclofen (n = 2, not shown). Hence a portion of the slow IPSP is likely to be attributable to non-GABAergic inhibition from GC1s. Representative examples are shown in Fig. 10. In one cell, tetanic stimulation produced a compound IPSP (Fig. 10A, left, right-arrow) that inhibited cell firing (Fig. 10, A, top 2 traces, and B, ). After bicuculline was applied, the IPSPs were blocked almost completely and the tetanic stimulus caused a much smaller reduction of the firing rate evoked by current injection. In another NBP cell (Fig. 10C), bicuculline substantially reduced the size of the IPSPs, but there remained a significant slow IPSP that resisted further attempts at antagonism, either with additional applications of bicuculline or with saclofen (data not shown). The reversal potential of this slow IPSP shifted from -78 to less than -85 mV after bicuculline, suggesting it was K+-mediated. This explains the generally very negative reversal potentials of the drug-naive slow IPSP: it is a mix of Cl--mediated responses (GABAA: reverses at -62 with exogenous GABA and -70 mV for the fast IPSP) and K+-mediated (unknown transmitter, likely GC1 in origin) responses.


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  		FIG. 10. Effect of GABAA antagonist bicuculline on NBP cell IPSPs. A: control responses (left) of a NBP cell to tetanic stimulation of DFL (200 Hz) were obtained during current injection (0.45-, 0.15-, 0-, 0.15-, 0.3-, 0.45-, and 0.6-nA step pulses). - - -, resting membrane potential (-69 mV). right-arrow, strong compound IPSP. After bicuculline was applied to the slice surface and the responses retested (right), the compound IPSP was abolished except for a small component (0.3 nA trial, right-arrow). Effect of bicuculline on the capacity of the compound IPSPs to inhibit cell firing is measured from the traces obtained during depolarizing current injection (0.45 and 0.6 nA). Instantaneous firing rates during control and bicuculline conditions are plotted in B. Under control conditions (), the cell showed some adaptation to current injection (slow decline in firing rate over time). At the onset of the tetanic stimulus (t = 0), the evoked IPSP reduces the firing rate, strongly during 0.45-nA injection and weakly during 0.6-nA injection. After bicuculline application (- - -), the tetanic stimulus caused a much smaller reduction in firing rate (0.45 nA; compared with current injection alone, not shown). C: response of NBP to tetanic stimulation before and after bicuculline application; in this cell there was a substantial (~25%) component of the slow IPSP that was insensitive to bicuculline. Bicuculline caused the reversal potential of the slow IPSP to shift from -78 to less than -85 mV.

In some NBP cells (n = 3), not only were the IPSPs antagonized, but a depolarizing or excitatory component was evoked by DFL stimulation after bicuculline application. This took the form of a slow depolarization (Fig. 11; n = 2) or spikes activated at very short latency (formed part of the stimulus artifact, n = 1). The cell shown in Fig. 11 responded to DFL stimulation with IPSPs alone. The resting membrane potential was very negative (-74 mV), therefore the short-latency IPSP evoked by a single DFL stimulus was depolarizing (Fig. 11, inset, control trace). This depolarizing IPSP and the compound IPSP evoked by tetanic stimulation (Fig. 11A, control) were abolished completely by bicuculline (Fig. 11B, bicuculline). Under these conditions, the tetanic stimulus evoked a slowly depolarizing potential; this potential was clearly voltage sensitive. We attribute these responses to the electrotonic synapses made by dendrites of GC1 and GC2 cells onto NBP somata and somatic dendrites (Maler et al. 1981) and the voltage-sensitive persistent Na+ currents present in these cells (Mathieson and Maler 1988; Turner et al. 1994). The rarity of this response is presumably also due to the low density of these connections (Maler 1979) and the difficulty of activating them in the slice preparation. This interpretation must however still be experimentally confirmed.1


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  		FIG. 11. Effect of GABA antagonists on NBP cell IPSPs. A and B: GABAA antagonist bicuculline. A: cell responded to single-pulse stimulation with a reversed (depolarizing) IPSP (see inset, control trace) at rest (-74 mV). Current injection during control conditions shows the compound IPSP to reverse a few millivolts above resting potential (main plot, - - -, control traces on left). B: application of bicuculline not only completely abolished the compound IPSP but revealed a slow depolarizing potential. Note that bicuculline blocks the depolarizing IPSP in the single-stimulus pulse experiment (A, inset). C and D: GABAB antagonist saclofen. Cell displayed spontaneous oscillations. C: tetanic stimulation (200 Hz, ) produced a robust long-duration IPSP (control trials, left). D: after saclofen application, the IPSP showed a small reduction in peak amplitude and duration. Approximate resting potential was -70 mV (- - -).

It has been suggested that gap junction input from granule cells and disinhibition may be responsible for NBP cell ON responses (Maler et al. 1981; Shumway and Maler 1989); however, the response of NBP cells to DFL stimulation when inhibition was blocked was at best only weakly excitatory in our experiments. Our data predict that other excitatory inputs, perhaps descending feedback, must be contributing to the excitatory responses of NBP cells seen in vivo.

GABAB ANTAGONISM. The effects of GABAB antagonists (saclofen and phaclofen) were tested on IPSPs evoked in NBP cells (n = 7). Only one cell showed any sensitivity to the antagonists, and even then the effect was marginal (Fig. 11, C and D). The tetanic stimulus produced a large compound IPSP. Several trials are shown to indicate the variability in the response. After 2 mM saclofen application, the IPSP showed a small reduction in duration. In all other NBP cells tested, the antagonists failed to significantly alter the time course or amplitude of IPSPs evoked by tetanic or single stimuli. We conclude that there is no significant GABAB component to the NBP cell IPSPs evoked via primary afferent stimulation.

EFFECT OF EXCITATORY ANTAGONISTS ON NBP CELL INHIBITION. The effect of blockade of excitatory-amino-acid (EAA) transmission was tested on NBP cell tetanus-evoked IPSPs. Non-NMDA (CNQX) and NMDA receptor antagonists (CPP or APV) were applied to the slice surface. The NBP cell in Fig. 12 responded to tetanic stimulation of the DFL with a strong compound IPSP. After CNQX and APV (1 mM) application, the IPSP mostly was abolished; during the stimulus period (Fig. 12, right, ), there remained a small inhibitory effect on current-evoked spiking. CNQX, with or without CPP/APV, reduced (n = 3) or eliminated (n = 2) the IPSPs produced by tetanic stimulation. Hence the inhibition evoked via primary afferent stimulation is mostly due to EAA-mediated synaptic activation of granule cell interneurons.


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  		FIG. 12. Effects of excitatory-amino-acid (EAA) transmission blockade on NBP cell IPSPs. Tetanic stimulation () produced strong long-duration IPSPs in the recorded cell. Under control conditions (left), current injection (-0.75-1.0 nA, 0.25-nA increments) reversed the polarity of the IPSP during the initial portion of the stimulus; reversal potential just below rest (-72 mV, - - -). After 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) + APV application, a small inhibitory effect was left during the tetanic stimulus. This inhibitory effect was only apparent during current injection (1 and 1.25 nA) as a small reduction in spike firing frequency during the tetanic stimulus. CNQX + APV also hyperpolarized the cell by 2 mV and reduced its input resistance; note the smaller voltage deflections evoked by the current steps.

Electrophysiological and pharmacological characteristics of synaptically evoked inhibition in BP cells

BP CELL INHIBITION. Electrophysiology. Cells were classified as BP cells if their response to DFL stimulation included a depolarizing and excitatory potential (EPSP, single stimulus) or a compound EPSP in their response to tetanic stimuli. As in NBP cells, tetanic stimulation was more effective than single stimuli at producing the strongest response. A representative range of responses to DFL stimulation is shown in Fig. 13.


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  		FIG. 13. Responses to single and tetanic stimulation of the DFL in 3 BP cells. Responses ranged from excitatory to mixed excitatory and inhibitory. A: BP cell with mostly excitatory response. A1: DFL stimulation (single stimulus) evokes EPSP with 2 peaks; at 1 and 4.8 ms. A2: current-clamp experiment (0.25-nA increments and as indicated) shows the voltage sensitivity of the EPSPs (-0.5-nA trace has the largest amplitude EPSP). A small slow IPSP appears to be present on the depolarized trace (sag toward end of response to current step), but it cannot prevent spiking in the more depolarized trials (0.5 and 0.75 nA). A3: response of same cell to a 200-Hz tetanic stimulus (black-triangle). Several spikes are evoked during the stimulus, whereas a single stimulus could not generate a spike (A1). There is no evidence of IPSPs. B: BP cell responded with similar EPSPs (see inset, 5-trial average) as the cell in A to a single stimulus; however, this cell also showed a slow (400-ms duration) IPSP after the EPSPs (single, 5-trial average). Tetanic stimulation (3-trial average) produced a mixed response with the EPSPs summating at first but then were overwhelmed by a strong IPSP(s) that also persisted for 400 ms. C1: BP cell responding to single stimulus mostly with an EPSP followed by a fast (peak at 2.5 ms) and slow IPSP (peak at ~260 ms). In some trials a spike was evoked by the stimulus (1 trial shown). C2: tetanic stimulation at 200 Hz but with only 5 stimulus pulses produced a mixed EPSP/IPSP response, with the inhibitory effect dominating; current clamp (rest, -0.25 and -0.5 nA) showed the reversal of the IPSPs just below resting potential (-71 mV), which prevents the EPSPs from reaching threshold. C3: net effect of the EPSP/IPSP mix is inhibitory as the tetanic stimulus (black-triangle, 200 Hz, 5 pulses) interrupts a current-evoked spike train (0.5 nA). C4: tetanic stimulation of 30 pulses at 200 Hz produces a summating EPSP that overcomes the inhibition to generate 4 spikes followed by a slow IPSP.

The cell in Fig. 13A (1-3) responded mostly with excitation to both single (with a bimodal EPSP) and tetanic stimulation. The tetanic stimulation was most effective, evoking several spikes (Fig. 13A3). However, in most cells, IPSPs also were evoked by single and tetanic stimulation (e.g., Fig. 13, B and C). The IPSPs appeared to curtail the summating EPSPs (e.g., Fig. 13B), resulting in a net hyperpolarization. The interaction of EPSPs and IPSPs (clearly separable in the response to single pulses, Fig. 13C1) could produce quite complex responses. For instance, in the cell in Fig. 13 C, a short tetanus of five pulses had a net inhibitory effect (Fig. 13C, 2 and 3) that prevented cell spiking, whereas tetani with more than five pulses (Fig. 13C4) were strongly excitatory.

In cells in which a single EPSP could be discerned after a single DFL stimulus, the first EPSP peaked at 1.2 ± 0.8 ms (n = 50). This was followed by a decaying depolarization (27 cells), which in some cells had a clear later EPSP peak at 18.8 ± 22.4 ms (n = 14) or by a clear early IPSP after the EPSP (23 cells), similar to the cell in Fig. 13C1. These early IPSPs peaked at 8.7 ± 7.6 ms (n = 6) and reversed at 71.7 ± 8.4 mV (n = 8). Slow IPSPs, such as those in the cells in Fig. 13, B and C, peaked at 197 ± 102 ms (n = 17) and reversed at -88.5 ± 0.5.2, with a mean duration of 813 ± 396 ms (n = 15). Compared with NBP cells (see Electrophysiological and pharmacological characteristics of synaptically evoked inhibition in NBP cells), BP-cell IPSP characteristics (early and late IPSPs) were not significantly different except for the slow IPSP duration, which was significantly shorter in NBP cells (NBP cell slow IPSP duration was 501 ± 371 ms, n = 17, P < 0.05). To determine whether the BP-cell IPSPs evoked by DFL stimulation showed pharmacological sensitivities similar to NBP cell IPSPs, the effects of GABA antagonists and EAA antagonists were tested on the BP-cell responses.

Pharmacology. 1) GABAA antagonism. The effects of GABAA antagonists were tested on the response of 10 BP cells to DFL stimulation. All of these cells showed varying degrees of mixed excitatory and inhibitory responses to DFL stimulation (see above). The example in Fig. 14 shows a typical effect of bicuculline. This BP cell responded to single-pulse stimulation of DFL with a large EPSP, followed by a fast IPSP (Fig. 14A), whereas tetanic stimulation (Fig. 14B) evoked a mixed excitatory and inhibitory response. Bicuculline completely blocked the fast IPSP (Fig. 14A) and the inhibitory component of the mixed response (Fig. 14B). It is noteworthy that the peak of the EPSP is unaffected by bicuculline, suggesting that the IPSP is delayed so as to overlap only the slower phase of the EPSP (see Berman and Maler 1998b for discussion of EPSP/IPSP timing). The small slow depolarizing response that followed the tetanus at rest (Fig. 14B, left) increased in magnitude after the bicuculline treatment.


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  		FIG. 14. Effect of bicuculline on a BP cell response to DFL stimulation. A: response to a single DFL stimulus before (control) and after (bicuculline) bicuculline application to the slice surface. EPSP is unaffected, whereas the IPSP is abolished completely by the drug, revealing a slow depolarizing potential. Note that in the control trace, the IPSP was followed by a slow depolarizing potential. Stimulus artifact blanked. B: responses to tetanic (200 Hz, 10 pulses) stimulation under current-clamp conditions (amplitude indicated, depolarized traces offset for clarity) before (control) and after (bicuculline) drug application. In the control case, the stimulus produced a mixed EPSP/IPSP response that was mostly inhibitory; only 2 spikes occur during the stimulus () despite the strong spiking evoked by the 0.75-nA current. At rest (-75 mV, - - -), a slow depolarizing potential is evoked by the tetanic stimulus. After bicuculline, the response to tetanic stimulation was purely excitatory. Spikes were evoked during the stimulus in all depolarized traces. A pronounced depolarizing potential followed the stimulus.

In all 10 cells GABAA antagonists increased the cells' net excitatory response to DFL stimulation. The nature of the effect depended on whether there was clear evidence of fast and slow IPSPs. Where fast IPSPs followed a DFL-evoked EPSP (n = 4), they were blocked completely by GABAA antagonists (e.g., Fig. 14A). Long-lasting IPSPs evoked by single or tetanic stimulation were blocked partially by GABAA antagonists (4 of 4 cells). Slow EPSPs (e.g., Fig. 14B), present in three BP cells, were augmented by GABAA antagonism. Two of these cells responded with a fast EPSP without evidence of a fast IPSP; bicuculline increased the amplitude of the fast EPSP, suggesting the presence of a masked IPSP. Hence in BP cells, primary afferent-evoked EPSPs always will be followed immediately by a fast GABAA IPSP.

2) GABAB antagonism. Unlike NBP cells, BP cells did have a saclofen/phaclofen-sensitive component of the slow IPSP. Figure 15 shows one such cell in which single-pulse stimulation evoked a EPSP followed by IPSPs (Fig. 15A). Tetanic stimulation evoked a mixed EPSP/IPSP where the EPSP had a short-lived effect on cell excitability; the summating IPSP quickly gained control over the cell after only two pulses of the stimulus (Fig. 15B, note the spike on the 1st pulse in the tetanus). Application of saclofen partially antagonized the slow component of the IPSP but had no effect on the early component. Of five BP cells tested, saclofen or phaclofen completely blocked (n = 3) or reduced the amplitude (n = 2) of the slow IPSP. Two additional BP cells were exposed to both bicuculline and saclofen; the effects of the two drugs were additive but failed to fully block the slow IPSP.