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J Neurophysiol 91: 2551-2567, 2004. First published January 28, 2004; doi:10.1152/jn.01121.2003
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Intracortical Pathways Determine Breadth of Subthreshold Frequency Receptive Fields in Primary Auditory Cortex

Simranjit Kaur, Ronit Lazar and Raju Metherate

Department of Neurobiology and Behavior, University of California, Irvine, California 92697

Submitted 20 November 2003; accepted in final form 20 January 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
To examine the basis of frequency receptive fields in auditory cortex (ACx), we have recorded intracellular (whole cell) and extracellular (local field potential, LFP) responses to tones in anesthetized rats. Frequency receptive fields derived from excitatory postsynaptic potentials (EPSPs) and LFPs from the same location resembled each other in terms of characteristic frequency (CF) and breadth of tuning, suggesting that LFPs reflect local synaptic (including subthreshold) activity. Subthreshold EPSP and LFP receptive fields were remarkably broad, often spanning five octaves (the maximum tested) at moderate intensities (40–50 dB above threshold). To identify receptive-field features that are generated intracortically, we microinjected the GABAA receptor agonist muscimol (0.2–5.1 mM, 1–5 µl) into ACx. Muscimol dramatically reduced LFP amplitude and reduced receptive-field bandwidth, implicating intracortical contributions to these features but had lesser effects on CF response threshold or onset latency, suggesting minimal loss of thalamocortical input. Reversal of muscimol's inhibition preferentially at the recording site by diffusion from the recording pipette of the GABAA receptor antagonist picrotoxin (0.01–100 µM) disinhibited responses to CF stimuli more than responses to spectrally distant, non-CF stimuli. We propose that thalamocortical and intracortical pathways preferentially contribute to responses evoked by CF and non-CF stimuli, respectively, and that intracortical projections linking frequency representations determine the breadth of receptive fields in primary ACx. Broad, subthreshold receptive fields may distinguish ACx from subcortical auditory relay nuclei, promote integrated responses to spectrotemporally complex stimuli, and provide a substrate for plasticity of cortical receptive fields and maps.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Prominent physiological features of primary auditory cortex (ACx) include short-latency neural responses to characteristic frequency (CF) stimuli, narrow frequency receptive fields (reflected in narrow threshold-tuning functions and response areas), and a topographic arrangement of CF representations (Doron et al. 2002Go; Merzenich et al. 1975Go; Phillips et al. 1985bGo; Sally and Kelly 1988Go). Similar features are found throughout the lemniscal auditory system (Calford et al. 1983Go), including in the ventral division of the medial geniculate thalamus (MGv), which provides the main auditory input to primary ACx (Roger and Arnault 1989Go; Romanski and LeDoux 1993Go). Because MGv neurons project to cortical neurons with the same CF (Imig and Morel 1984Go; Miller et al. 2001Go; Winer et al. 1999Go), it is plausible that response characteristics exhibited by cortical neurons simply reflect response characteristics of the afferent thalamic neurons. Similarly, it might seem unlikely that response features in ACx that resemble those seen throughout the lemniscal auditory pathway would reflect a cortical specialization.

Breadth of tuning may be a notable exception. Frequency receptive fields typically are determined using extracellular recordings of spike discharge in response to pure tone stimuli. However, the narrow receptive fields thus derived may be misleading and underestimate the spectral breadth of inputs to cortical neurons as evidenced by a number of studies. Blockade of intracortical inhibition results in an expansion of frequency receptive fields derived from extracellular spike discharge, suggesting the presence of normally subthreshold excitatory postsynaptic potentials (EPSPs) that are inhibited by intracortical circuits (Foeller et al. 2001Go; Muller and Scheich 1988Go; Wang et al. 2000Go, 2002Go). Also, direct, intracellular recordings of synaptic inputs reveal that subthreshold receptive fields extend beyond the boundaries of suprathreshold (derived from spikes) receptive fields (Ojima and Murakami 2002Go; Ribaupierre et al. 1972Go; Volkov and Galazjuk 1991Go; Wehr and Zador 2003Go). Subthreshold receptive fields (sometimes referred to as subliminal, surround, or nonclassical receptive fields) could contribute to ACx function in a variety of ways. For example, EPSPs that are subthreshold when evoked by pure tones could integrate, spatially and temporally, with other EPSPs elicited by spectrotemporally complex stimuli to elicit spikes. Further, in aroused or attentive animals or animals undergoing behavioral training with acoustic stimuli, receptive fields could be larger, or differently shaped, than those observed under anesthesia (Edeline et al. 2001Go; Weinberger and Bakin 1998Go). Finally, changes in synaptic strength of previously subthreshold inputs by behavioral (or other) manipulations could underlie large scale reorganization of frequency representations in ACx (Kilgard and Merzenich 1998Go; Recanzone et al. 1993Go).

The present study was designed to answer two questions central to the issue of subthreshold receptive fields in ACx: how broad are they and to what extent do they involve integration of thalamocortical and intracortical inputs? We made intracellular and local field potential (LFP) recordings of toneevoked responses to measure synaptic activity, focusing on the initial, presumed excitatory, components. We determined onset latencies for responses to CF and non-CF stimuli to infer functional connectivity from the arrival time of acoustic inputs. We then determined the effect of intracortical muscimol microinjections on receptive field bandwidth because this manipulation should preferentially inhibit cortical (vs. thalamocortical) neurons. And finally, we reversed the effect of intracortical muscimol preferentially at the recording site (within a larger muscimol-inhibited cortical region) to distinguish between two hypotheses of the functional connectivity underlying receptive fields. The results indicate that at a given cortical site, thalamocortical and intracortical pathways preferentially mediate responses to CF and non-CF stimuli, respectively.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Surgical procedure

All procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of California, Irvine IACUC. Male Sprague-Dawley rats (Charles River Laboratories, Hollister, CA), age >1 mo and weighing 110–240 g were used for combined intracellular and LFP studies. Adult rats weighing 250–500 g were used for LFP-only studies. Animals were anesthetized with 1.5 g/kg ip urethan (Sigma, St. Louis, MO) and 10 mg/kg ip xylazine (Phoenix Pharmaceuticals, St. Joseph, MO) and subsequently administered 0.6 mg/kg ip atropine (Phoenix). Animals remained in a state of deep anesthesia throughout the surgery and were given supplemental injections of urethan (40 mg) and xylazine (0.25 mg) at ~2-h intervals to maintain areflexia and a synchronized cortical electroencephalogram (EEG). Body temperature was maintained at 36–37° C using a feedback-controlled heating pad (Harvard Instruments, Holliston, MA). The animal was placed in a sound-attenuating chamber (model AC-3, IAC, Bronx, NY) mounted on an air table (Newport, Irvine, CA), and the head was secured in a stereotaxic frame (model 923, Kopf Instruments, Tujunga, CA) using blunt earbars (Kopf). A midline incision was made, and xylocaine ointment (AstraUSA, Westborough, MA) was applied to the incision. After the skull was cleared, the head was secured by a custom-made head holder screwed and cemented onto the skull. A craniotomy was performed over the right ACx, and the exposed cortex was kept moist with warmed saline. The earbars were removed. In early experiments, a drawing of the surface vasculature helped with reconstruction of recording sites. In most experiments, a Polaroid picture was taken of the exposed cortex through the surgical microscope (Carl Zeiss, Thornwood, NY) for the same purpose. To improve stability during experiments that included intracellular recordings, tracheal cannulation, lumbar suspension, and drainage of the cisterna magna were performed in addition to the surgery described in the preceding text. These procedures helped minimize brain pulsations caused by blood pressure fluctuations and respiration. After the experiments animals were killed with a lethal dose of anesthesia.

Electrophysiology

Primary ACx in the rat lies on the dorsolateral aspect of temporal cortex, framed by a characteristic blood vessel pattern (Sally and Kelly 1988Go). Recording from the cortical surface within this area, we used short-latency, large-amplitude responses to click stimuli to localize ACx (Barth and Di 1990Go; Shaw 1988Go) and then made multiple penetrations into layer 4 of the cortex to confirm the tonotopic arrangement characteristic of primary ACx (Doron et al. 2002Go; Horikawa et al. 1988Go; Kilgard and Merzenich 1998Go; Sally and Kelly 1988Go).

INTRACELLULAR RECORDING. Methods for in vivo whole cell recordings are similar to those described previously (Metherate and Ashe 1993aGo). Patch pipettes were pulled from glass (1.5 mm OD glass, A-M Systems, Carlsborg, WA) on a horizontal puller (P-97, Sutter Instruments, Novato, CA) and had a tip diameter of ~2.5 µm and resistances ranging from 8 to 14 M{Omega} when filled with (in mM) 140 K+-gluconate, 1 MgCl, 1 CaCl, 10 HEPES, and 1.6 EGTA, adjusted to pH 7.3 with KOH. The osmolality of the recording solution was adjusted to ~260 mmol/kg. All drugs and chemicals were obtained from Fisher (Fair Lawn, NJ), with the exception of D-gluconic acid, which was obtained from Sigma Chemical.

Whole cell recordings were made from neurons in layers 2–4 as described previously (Blanton et al. 1989Go; Metherate and Ashe 1993aGo). Briefly, the electrode was inserted perpendicular to the pial surface, lowered to a depth of ~200 µm using a microdrive (Inchworm, Burleigh Instruments, Fishers, NY) and weak positive pressure applied to avoid clogging the electrode tip [positive or negative pressure was applied through the side-port of the sealed electrode holder (Warner Instruments, Hamden, CT)]. The positive pressure was then removed and the electrode was advanced slowly with a current pulse (100 pA, 30 ms) delivered once per second. Proximity to a neuron's membrane produced an increased voltage response to the current pulse, at which time the application of a small amount of negative pressure resulted, ideally, in an increased voltage response that eventually indicated a tight seal (~1G{Omega}). When a stable gigaohm seal was achieved, steadily increasing negative pressure was applied to rupture the membrane. Occasionally, the membrane ruptured spontaneously. A successful rupture was indicated by the sudden appearance on the oscilloscope of a membrane potential (approximately –70 mV) with rhythmic voltage fluctuations (Metherate and Ashe 1993aGo).

Responses to acoustic stimuli were recorded using an intracellular amplifier (Axoclamp 2B, Axon Instruments, Foster City, CA), viewed on a digital oscilloscope (Tektronix, Irvine, CA), digitized at 5 kHz (IT-16, Instrutech, Port Washington, NY), averaged (20 or 40 trials), and stored on a computer (Macintosh G4, Apple Computer). Data acquisition was triggered 100 ms before acoustic stimulation. Computer software (AxoGraph, Axon Instruments) controlled data acquisition and analysis.

EXTRACELLULAR RECORDING. LFP recordings were obtained in nearly all experiments using glass microelectrodes (1.5 mm OD glass, A-M Systems; tip diameter: ~2.5 µm, filled with 1 M NaCl, ~1 M{Omega} impedance at 1 kHz) pulled in multiple stages to obtain blunt tips, as for patch pipettes. In two experiments, dual tungsten electrodes were used for simultaneous recording from two sites (1–2 mm separation). Electrodes were inserted perpendicular to the pial surface, and movement was controlled using a microdrive (Burleigh Inchworm). Neural activity was filtered and amplified (1 Hz to 10 kHz, AI-401 Cyber-Amp, Axon Instruments) displayed on the oscilloscope, digitized at 5 kHz, and stored on computer. Data acquisition was triggered 100 ms before acoustic stimulation, and responses were averaged (25 or 50 trials; AxoGraph, Axon Instruments). The local EEG was monitored continuously on the oscilloscope.

Acoustic stimulation

Pure tone stimuli were digitally synthesized and controlled using MALab (Kaiser Instruments, Irvine, CA) and a dedicated computer (Macintosh PowerPC, Apple Computer) and delivered through an electrostatic speaker (ES-1 with ED-1 driver, Tucker-Davis Technologies, Gainesville, FL) positioned ~3 cm in front of the left ear. For calibration (SPL in decibels re: 20 µPa), a microphone (No. 4939 microphone and Nexus amplifier; Bruel and Kjaer, Norcross, GA) was positioned in place of the animal at the tip of the left earbar. Tones were 100 ms in duration with 10-ms linear rise and fall ramps.

Pharmacological manipulations

To inhibit intracortical activity, muscimol hydrobromide (5.1 mM in saline; Sigma) was administered through a glass micropipette with a broken tip (~20–30 µm diam) attached via polyethylene tubing to a 1 or 5 µl Hamilton syringe (WPI, Sarasota, FL). The pipette was inserted into the cortex at the recording site after removal of the recording electrode. Muscimol (or saline, for controls) was injected in increments of 0.5–1.0 µl over 1–2 min, or, in one early experiment, muscimol was applied to the cortical surface. The muscimol pipette was left in place for 15 min after the injection and then removed and replaced with the recording electrode, after which tone-evoked responses were obtained. In some cases, this procedure was repeated one or more times to increase the muscimol dose. In later experiments, to investigate reversal of inhibition by picrotoxin (0.01–100 µM in saline; Sigma), we used a lower concentration of muscimol (200 µM, 0.5–2.0 µl). After recording responses in muscimol-inhibited cortex, the recording electrode was replaced with one containing picrotoxin.

Analysis of acoustic-evoked responses

CF and other acoustic response features were determined for a particular recording site by examining LFPs evoked by a standard stimulus set of six frequencies spanning five octaves (1 or 1.25 to 40 kHz in ~1-octave steps) delivered at intensities from 70 dB SPL to below CF threshold, typically in 10 dB steps. Although the 1 octave resolution of the stimulus set is relatively crude, it was sufficient to determine changes in tonotopic organization over the cortical distances examined (~1 mm steps), and we use the conventional term "CF" for convenience. CF was determined qualitatively by identifying the frequency of the stimulus with the lowest threshold response. When stimuli of more than one frequency elicited a response at threshold, intensity was varied by 5 dB and/or CF was determined by the response with the shortest onset latency (e.g., Fig. 3A, responses at 0 dB). Quantitative measures of evoked intracellular and LFP onset latencies (see details in the following text) and peak amplitudes were obtained for all responses. For measurements of response amplitude, baseline was defined as the average potential from tone onset to 5 ms after stimulus onset.



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FIG. 3. Features of tone-evoked LFPs in layer 4 of mid- and high-frequency cortical sites separated by ~2 mm. A and B: example responses from mid- and high-frequency sites in the same animal. Only partial response sets are shown to demonstrate relevant features; tone onset is at beginning of each 50 ms trace. {uparrow}, in this and subsequent figures, threshold for CF stimulus-evoked response. C: onset latency data averaged from mid- and high-frequency sites in 19 animals, separately for each stimulus intensity (10–60 dB, relative to minimum threshold at each site). D: onset latency for CF stimulus-evoked response at high-frequency site was consistently less than at mid-frequency site (paired t-test with all intensities grouped, n = 17, P < 0.001). For C and D, data points are averages (±SE) from >= 3 animals. ANOVAs for each intensity level in C revealed significant differences for 10 and 30–60 dB at the mid-frequency site (P < 0.01), and 10–60 dB at the high-frequency site (P < 0.05).

 
We defined a "response" at each recording site as a voltage deflection exceeding 2 SDs from the mean baseline obtained from all responses to the standard stimulus set (typically 48 responses: 6 frequencies x 8 intensities). Two additional criteria were helpful near threshold: a voltage >2 SDs was considered a response only when there were clear responses to the same frequency stimulus at higher intensities and the onset latency was equal to or greater than the latency at higher intensities.

A major part of the present study involved obtaining objective and accurate estimates of response onset latencies. The method is illustrated in Fig. 2B for an LFP. First, a "threshold" 1 SE below the mean baseline was established, and the point at which the response crossed the threshold determined. Then, two points 1 ms before and after the threshold-crossing were identified, and the slope of the line connecting these two points was determined. Finally, the intersection of this line with the average baseline potential was obtained and defined as response onset (Fig. 2B, small arrow). A similar procedure was used to obtain the onset latencies of intracellular responses.



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FIG. 2. EPSP and LFP onset latencies increase with spectral "distance" from CF. A: superimposed LFP (top) and intracellular (bottom) responses show increase in onset latency with increasing spectral distance from CF (same data as in Fig. 1A, 70 dB; the 5-kHz stimulus-evoked LFP response had an onset latency similar to that for 20 kHz, but the trace crossed other responses and was removed for clarity). B: sample LFP response to illustrate features measured quantitatively (see METHODS). C: average intracellular onset latencies show trend toward longer onset latencies with increasing spectral distance from CF despite increased variability associated with more spectrally distant means (n = 3–8 neurons for each mean value). Only latencies at –1 octave were significantly longer than at CF (1-tailed paired t-test, n = 7–8, P < 0.05).

 
Statistical analyses

Statistical analyses were performed using Statview (v. 4.5 for Macintosh, Abacus Concepts). Tests on independent means were Student's t-test and factorial ANOVA, whereas the paired t-test and repeated-measures ANOVA were used for related means. In analyzing response features obtained at different frequencies and intensities (e.g., Fig. 3C), repeated-measures ANOVAs were performed separately at each intensity; for such ANOVAs, only a subset of the data was included so that the mean values being compared contained equal n (however, all data are plotted in figures). Detailed statistics results are in the figure legends; P < 0.05 was considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
We have investigated the functional connectivity underlying frequency receptive fields in primary ACx by recording tone-evoked, synaptic responses (intracellular recordings) and presumed synaptic responses (LFPs) in 50 rats. The data comprise intracellular and LFP responses to a standard set of six frequencies (1 or 1.25, 2 or 2.5, 5, 10, 20, and 40 kHz) spanning five octaves at up to nine stimulus intensities. We also employed pharmacological manipulations designed to distinguish between thalamocortical and long-distance, intracortical connectivity. Although the latter portions of the study utilize only LFP recordings, we first describe intracellular synaptic responses to acoustic stimuli obtained in whole cell recordings and compare them to LFP recordings obtained at the same location to confirm that LFP characteristics reflect those of known synaptic responses.

Intracellular recordings in primary ACx

We obtained intracellular recordings from eight neurons in primary ACx, using the in vivo whole cell recording method (Metherate and Ashe 1993aGo). Cell depths ranged from 275 to 612 µm below the pia, corresponding to layers 2–4. In cell-attached mode, the seal resistance was 1.0 ± 0.1 G{Omega}; after rupture of the membrane with suction, the resting membrane potential (RMP) was –69 ± 3.2 mV, and the input resistance, estimated from responses to small hyperpolarizing current pulses, was 182 ± 64.2 M{Omega}. The duration of recording ranged from 9 to 47 min.

Example intracellular responses to acoustic stimulation are shown in Fig. 1A (top), for a layer 3 neuron. For this neuron, rupture of the cell membrane was obtained using minimal suction after the seal resistance had reached a plateau at 1.5 G{Omega}. After break-in, rhythmic membrane potential fluctuations characterized spontaneous activity as described previously (Metherate and Ashe 1993aGo; Metherate et al. 1992Go). The RMP was –81 mV immediately on break-in, but generally ranged from –52 to –63 mV for most of the recording (RMP defined as the potential during the peak hyperpolarizing phase of membrane potential fluctuations, in the absence of injected current). Spontaneous action potentials occurred during depolarizing phases of membrane potential fluctuations in about half the neurons. In most neurons, we made small adjustments in the level of injected DC current over time to maintain the membrane potential close to the RMP observed at the beginning of each data collection sequence. However, data obtained when the RMP was more depolarized than –50 mV were discarded.



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FIG. 1. Intracellular (whole cell) and local field potential (LFP) recordings from the same cortical location show similar characteristic frequency (CF) and breadth of tuning. A: intracellular responses from a layer 3 neuron (top) and LFP responses from the same layer 3 location (bottom) had a CF of 10 kHz as evident from responses to 20-dB stimuli. Responses to 70-dB stimuli demonstrated intracellular and LFP bandwidths >=5 octaves. Tone onset is at the beginning of each trace as indicated (schematic at bottom left; 10 ms ON-OFF ramp). B: scatterplot showing similar intracellular and LFP bandwidths at 10–60 dB above response threshold at CF (n = 1–5 neurons/extracellular sites at each intensity). ···, maximum detectable bandwidth, 5 octaves. C: paired intracellular and LFP bandwidths (means ± SE, same data as in B) at 10 and 20 dB above CF threshold did not differ [paired t-test, n = 4–5, P > 0.05; excitatory postsynaptic potential (EPSP) bandwidth (BW)10 (in octaves) = 1.25 ± 0.75, LFP BW10 = 2.2 ± 0.58; EPSP BW20 = 2.75 ± 0.85, LFP BW20 = 2.75 ± 0.63]. IC, intracellular.

 
Figure 1A (top) shows synaptic responses to the standard set of six frequencies at 20 and 70 dB SPL. At the lower intensity, only 10-kHz stimuli produced a clear, short latency depolarization; i.e., 10 kHz was CF. At the high intensity, stimuli of all frequencies elicited EPSPs, followed by apparent inhibitory postsynaptic potentials (IPSPs), which were more clearly visible at the edges of the receptive field (e.g., responses to 1.25 and 40 kHz in Fig. 1A). Note that in other neurons, IPSPs were not always visible at RMP as overt hyperpolarizations nor were they restricted to the receptive field edges. Also apparent in the figure is that EPSP onset latencies decreased with increasing stimulus intensity, and increased with increasing spectral distance from CF (see also Fig. 2, A and C).

The receptive field bandwidth derived from tone-evoked EPSPs was broad, measuring >=5 octaves (the maximum we could test) at 70 dB for the example in Fig. 1A. Across neurons, bandwidth ranged from 3 to >=5 octaves at moderate to high intensities (Fig. 1B). At 10 and 20 dB above threshold, where determination of bandwidth was not limited by the 5-octave stimulus range, EPSP bandwidth averaged 1.25 ± 0.75 and 2.75 ± 0.85 octaves, respectively (Fig. 1C). Although stimuli were delivered in relatively crude, 1 octave steps, these bandwidth estimates indicate remarkably broad synaptic receptive fields.

Tone-evoked EPSPs typically did not elicit spikes unless the neuron was depolarized with DC current; but in three neurons, higher-intensity stimuli did elicit spikes at RMP. Stimuli <=1 octave above CF (n = 2) or 2 octaves below CF (n = 1) produced suprathreshold responses, and spike-based bandwidth ranged from 1 to 2 octaves. In contrast, subthreshold bandwidth in these cells ranged from 3 to >=5 octaves. In other words, subthreshold receptive fields were much broader than spike-based receptive fields.

Comparing intracellular and LFP responses to pure tones

After each intracellular recording, LFPs were recorded at the same location to allow for a direct comparison of intracellular and LFP features (n = 7). LFPs were recorded using electrodes similar to patch pipettes but with slightly larger tips and correspondingly lower impedances (~1 M{Omega}). Figure 1A (bottom) shows an LFP recording from the same layer 3 site as the intracellular responses (top). Tone-evoked LFPs were invariably of negative polarity, as expected for extracellular responses near the site of excitatory synaptic activity (Barth and Di 1990Go; Metherate and Ashe 1993bGo; Metherate et al. 1992Go; Muller-Preuss and Mitzdorf 1984Go). As can be seen in Fig. 1A, several LFP features resemble those of intracellular responses: both sets of responses indicate a CF of 10 kHz (sole, or shortest-latency, response at 20 dB), a bandwidth >=5 octaves at 70 dB, and similar changes in onset latency with increasing intensity or spectal distance from CF (also see Fig. 2A).

Group data confirm the similarities between intracellular and LFP responses. CFs derived from intracellular and LFP responses were identical in 5/6 cases (in 1 cell, the intracellular CF could not be determined clearly). However, CF thresholds were higher in intracellular recordings (average: 40 ± 5.5 dB, range: 20–50 dB) compared with LFP recordings (average: 30 ± 5.5 dB, range: 10–40 dB; paired t-test, n = 5, P < 0.001). Bandwidths for paired intracellular and LFP recordings are shown in Fig. 1B and covered a similar range at each intensity up to the maximum detectable 5 octaves at high intensities. At 10 and 20 dB above threshold, where bandwidth determination was not limited by our stimulus range, intracellular and LFP bandwidth did not differ (Fig. 1C).

An important goal of this study was to infer functional connectivity, in part from differences in onset latency among LFP responses to stimuli of different frequencies. We therefore examined EPSP and LFP onset latencies and found that the response to CF stimuli had the shortest onset latency while the responses to non-CF stimuli— both higher and lower frequencies—were progressively longer with increasing spectral "distance" from CF. An example is shown in Fig. 2A, using intracellular and LFP recordings from the same site. For LFPs (Fig. 2A, top) and EPSPs (bottom), CF (10 kHz) stimuli elicited the shortest onset latency and responses to stimuli of other frequencies tended to have progressively longer latency onsets with increasing spectral distance from CF. Note that this trend holds for frequencies above and below CF; e.g., LFP and EPSP responses to stimulus frequencies 2 octaves above and below CF (40 and 2.5 kHz, respectively), all have longer latency onsets than do responses to stimuli at CF.

As can be seen for some traces in Fig. 2A, the exact timing of individual onsets was not always clear, especially when the onset was not abrupt. The difficulty in identifying onset precisely led us to devise an objective method for estimating onset latency. The method is illustrated in Fig. 2B for an LFP response and is detailed in METHODS. Briefly, onset latency was determined by extrapolating a line based on the slope of the LFP around a threshold point established from statistical variations (–1 SE) in the baseline potential. A threshold relatively near to the baseline was chosen because response onset often seemed to begin well before the strongest portion of the response, producing an initially gradual, two-component onset (Fig. 2B). A near-baseline threshold ensured that the early component of the response influenced our estimate of onset latency and resulted in more accurate estimates than if the strongest portion of the response was extrapolated to the baseline instead. Average LFP latencies are described in greater detail in the following text (Fig. 3C) and clearly show increasing onset latencies with spectral distance from CF.

Intracellular onset latencies were determined similarly. Onset latency data at 70 dB (~20–30 dB above threshold) showed that CF stimuli tended to have the shortest onset latency (average: 18.5 ± 1.53 ms, range: 12.9–26.8 ms) with responses to non-CF stimuli having progressively longer onset latencies with distance from CF. However, a wide range of response latencies resulted in limited statistical significance (Fig. 2C).

Taken together, the comparison of intracellular and LFP data in terms of polarity, bandwidth, and changes in onset latency with intensity and spectal distance from CF, demonstrate that LFPs reflect important features of local synaptic activity. Because LFPs are suitable for the long-duration recordings needed for this study, at a predetermined depth (layer 4) in each animal and before and after pharmacological manipulations, we subsequently focused on LFPs to infer synaptic connectivity in ACx.

Layer 4 LFP response to pure tones

In 25 animals, we recorded tone-evoked LFP activity in multiple penetrations across ACx. These multiple penetrations were performed to confirm the topographic arrangement of frequency representations expected for primary ACx and to determine which, if any, features of LFP responses varied across frequency representations (changes in response features observed in multiple penetrations across ACx also would support further the notion that LFPs reflect local activity). At each site, the recording depth was kept constant at 600 µm to sample activity in layer 4 (in 5 experiments, current source density analysis revealed the major short-latency, tone-evoked current sink to be at 600 µm; data not shown). To determine CF and other response features, we used the same standard, 5 octave stimulus set as before. In each animal, we first determined the area responsive to sound stimuli by recording surface responses to clicks. We made a first penetration near the anterior end of the shortest-latency, largest-amplitude surface responses (click map), and recorded layer 4 responses to the stimulus set. We then made one to four additional penetrations in ~1 mm steps directly posterior to the first penetration and recorded layer 4 responses to the same stimulus set. In most animals (n = 19), we observed a shift in effective stimuli from high frequencies at the anterior penetration to lower frequencies at more posterior locations (arrows in Fig. 3 and subsequent figures indicate CF threshold). This sequence is consistent with previous reports of the tonotopic organization of primary ACx in the rat (Doron et al. 2002Go; Horikawa et al. 1988Go; Sally and Kelly 1988Go).

In the 19 animals with a confirmed tonotopic arrangement, the region of the first penetration was invariably a high-frequency area with CF = 40 kHz (Fig. 3B). Because 40 kHz was the highest frequency delivered, these anterior sites might actually have higher CFs. However, the threshold intensity at these sites was relatively low, averaging 9.5 ± 1.6 dB SPL (range: 0–20 dB SPL), similar to thresholds seen in more posterior penetrations with lower frequency CFs (in the following text). Thus the anterior sites likely have CFs near 40 kHz. In each animal with a tonotopic arrangement, responses revealed a lower-frequency region at a second site more posterior to the "high-frequency site" (Fig. 3, A and B). This "mid-frequency site" was 1.5–2 mm posterior (mean: 1.7 ± 0.1 mm). CF at the mid-frequency site was usually 10 kHz (n = 15) but sometimes 5 kHz (n = 3) or 20 kHz (n = 1). Thresholds at the mid-frequency sites averaged 16.3 ± 3.3 dB SPL (range: 0–40 dB SPL) not different from those at the high-frequency site (paired t-test, n = 19, P > 0.05).

In 6/25 animals examined for frequency organization, no clear evidence for tonotopy emerged. In these animals, two to four penetrations were made spanning a distance of 0.8–2.8 mm (mean: 1.7 ± 0.3 mm) from the anterior portion of the click map, i.e., the same procedure that revealed tonotopy in most animals. Unlike in the animals with clear tonotopy, however, several sites <=2.8 mm apart appeared to have the same CF (n = 5), and/or some sites responded weakly at threshold to a range of frequencies spanning three octaves or more (n = 2). For the latter animals, small and variable responses near threshold prevented a clear determination of CF and therefore may have prevented determination of a progression of CFs with distance. However, CFs were clear in other cases that did not show a change of CF with distance. Further, responses at higher intensities were robust in all 6 animals, yet 5/6 did not show the expected shift of frequency ranges with distance (cf. responses at 30 dB SPL in Fig. 3, A and B, for expected shift). The surface blood vessel patterns and distribution of click responses in these animals appeared similar to tonotopic animals, suggesting that recordings were made in analogous cortical regions. However, absent a fine-grain mapping procedure, it remains possible that we were recording in a nonprimary field (Doron et al. 2002Go; Horikawa et al. 1988Go; Sally and Kelly 1988Go). In any case, data from animals lacking tonotopic organization were not analyzed further.

Features of tone-evoked responses that imply connectivity

For the 19 animals with confirmed recordings in tonotopic primary ACx, we undertook a detailed analysis of the layer 4 responses to the standard stimulus set. For each animal, data were analyzed both at the high-frequency site (CF = 40 kHz) and at the mid-frequency site (CF generally 10 kHz). We first analyzed the onset latency of tone-evoked LFP responses because minimum onset latencies at a particular site are determined, in part, by anatomical connections. As is clear from the LFP examples already described (Figs. 2A and 3, A and B), and from the mean LFP onset latencies in Fig. 3C, onset latency increased for stimulus frequencies away from CF at both high- and mid-frequency sites. Changes in onset latency were smaller, yet still significant, at higher intensities; e.g., for the mid-frequency site in Fig. 3C, changes in onset averaged 4.3 ms/octave at 10 dB and 0.7 ms/octave at 60 dB above threshold (data averaged in 1 octave intervals from –3 to +1 octaves from CF). Onset latencies for CF stimuli decreased with increasing intensity at each site (Fig. 3C, ANOVAs, P values <0.01) and appeared to asymptote at higher intensities (50–60 dB, Fig. 3D). Finally, for a given intensity, CF stimulus-evoked onset latencies at the high-frequency site were consistently less than those at the mid-frequency site (Fig. 3D).

Note, as previously mentioned (Fig. 1), that LFP responses at moderate to high intensities could span 5 octaves. Even more remarkably, such data at a high-frequency site indicate a receptive field extending at least five octaves below CF (for an individual example, see Fig. 4C). These broad receptive fields are similar to the subthreshold receptive fields seen in intracellular recordings (Fig. 1) but substantially broader than those derived from suprathreshold (single unit) activity in primary ACx (see DISCUSSION).



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FIG. 4. "Boosted" responses elicited by below-CF stimuli at higher intensities. A–C: all data from the same animal; tone onset at start of traces. Isointensity responses boxed in A are superimposed in B with amplitudes normalized to facilitate comparison of onset latencies. D: intracellular responses to 5 kHz (CF) and 1.25 kHz stimuli at 70 dB showing a boosted response to 1.25 kHz stimulus but shorter-latency response to CF stimulus. E: peak amplitude plotted vs. spectral distance from CF at different intensites. Data from 19 animals, each data point is average (±SE) from >=3 animals. ANOVAs for each intensity level revealed no significant differences at the mid-frequency site and significant differences at 10–60 dB at the high-frequency site (P < 0.01)

 
Measurements of LFP peak amplitude (Fig. 2B) often showed the expected decreased amplitudes at stimulus frequencies away from CF (e.g., Fig. 3, A and B). However, a more striking—and unexpected—feature observed in many animals was an increased amplitude for stimulus frequencies below CF, especially at high intensities (Fig. 4). Examples of this "boosting" of response amplitudes to below-CF stimuli are shown in Fig. 4, A and C (boosting defined as response amplitudes to non-CF stimuli greater than amplitudes to CF stimuli). Prominent boosting occurred in 19/38 sites in 14/19 animals and always at frequencies below CF (Fig. 4E). Boosting also occurred in intracellular recordings, in 2/8 cells, at high intensity (Fig. 4D). Note in the example (Fig. 4, A and C) and the mean data (Fig. 4E) that boosting is seen for stimuli <=3 octaves below CF at both midand high-frequency sites. Note, also, that despite the boosting of responses to non-CF stimuli, the response to CF stimuli still had the shortest onset latency (Fig. 4B; peak amplitudes normalized to facilitate comparison of onsets). Intracellular recordings confirmed shorter onset latencies for CF stimulus-evoked EPSPs despite boosting of responses to non-CF stimuli (Fig. 4D). The effects of boosting dominated the average data at higher intensities for stimulus frequencies below CF (Fig. 4E). Response amplitudes to CF stimuli increased monotonically, on average, with increasing stimulus intensity, even when analyzing data only from sites displaying boosted responses.

Two hypotheses on the connectivity underlying frequency receptive fields

LFP features have important implications for how frequency receptive fields are constructed in ACx. For example, at a given recording site, CF stimuli elicit the shortest latency responses, implying a direct anatomical connection from the thalamus. Non-CF stimuli—<=5 octaves from CF— elicit longer-latency responses. Two hypotheses posit extreme scenarios to account for how CF and non-CF information arrive at a given recording site (Fig. 5; for this exercise, we loosely define "non-CF" as several octaves from CF). In the first (Fig. 5A), extensive thalamocortical terminal arbors project over a wide cortical area and thus relay non-CF as well as CF information to the recording site. According to this scheme, increased latencies for non-CF input could result from longer thalamocortical path lengths. In the second scenario (Fig. 5B), non-CF information arrives at the recording site via an intracortical mono- or polysynaptic pathway. The increased latency for non-CF input therefore results from intracortical processing that includes one or more synaptic delays. (An additional factor, that non-CF stimuli elicit longer latency spikes than CF stimuli throughout the auditory system, applies to both hypotheses but does not account for the small timing differences among synaptic onsets observed here; see DISCUSSION). The remainder of this study is aimed at distinguishing between these two hypotheses.



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FIG. 5. Two alternative hypotheses of thalamocortical and intracortical connectivity that could give rise to broad frequency tuning and increasing onset latencies with spectral distance from CF. A: thalamocortical arbors are extensive and relay both CF and non-CF information from the thalamus directly to the recording site. B: CF information arrives at the recording site via thalamocortical projections, whereas non-CF information is relayed by intracortical projections.

 
Inhibition of intracortical activity via muscimol microinjection into ACx

To examine the involvement of intracortical processing in relaying CF versus non-CF inputs, we inhibited intracortical activity with microinjections of muscimol, a GABAA receptor agonist that inhibits postsynaptic activity but not fiber activity (for review, see Martin and Ghez 1999Go).

In nine experiments, after recording baseline responses we applied muscimol (5.1 mM; 1–2.5 µl) to the mid-frequency recording site. In four of these nine experiments, muscimol was microinjected (1.0 µl) into layer 4. In one experiment, muscimol was applied to the cortical surface. In another four experiments, we made two to five injections into layer 4, typically 0.5 µl per injection, 30–60 min apart with intervening recordings that revealed progressively stronger suppression, and we assumed cumulative doses because the effects of muscimol at this concentration are long-lasting (Edeline et al. 2002Go; Martin and Ghez 1999Go; present study). Muscimol at such doses would be expected to diffuse several millimeters from the injection site (Edeline et al. 2002Go; Martin and Ghez 1999Go) and inhibit much of primary ACx. We began data collection ~15–20 min after the muscimol injection(s). Muscimol strongly reduced CF stimulus-evoked LFP magnitude for hours (Fig. 6A; recovery was attempted in 1 case, with ~50% recovery seen after 6 h). On average, muscimol significantly reduced response peak amplitude for intensities at and above threshold (Fig. 6B) and reduced completely the later components of evoked responses (Fig. 6A). In 6/9 animals, threshold either did not change (Fig. 6A, arrowhead indicates predrug CF threshold) or increased 10 dB. In 3/9 animals, threshold after muscimol increased 20–40 dB. Nonetheless, muscimol did not significantly change average CF threshold (2.2 ± 2.22 dB before muscimol vs. 13.3 ± 4.71 dB after; paired t-test, n = 9 pairs, P = 0.062). As a control for nonspecific effects, injection of 0.5 µl (n = 1 animal) or 1 µl (n = 4) saline at the recording site did not affect responses to CF stimuli (93.1 ± 6.2% of predrug amplitude, all intensities pooled; Fig. 6B), whereas 1 µl muscimol reduced amplitudes to 25.7 ± 2.1% of baseline (paired t-test with all intensities pooled, P < 0.05). Saline also did not affect response threshold (predrug threshold: 4.0 ± 2.45 vs. 4.0 ± 2.45 dB after saline; paired t-test, n = 5 pairs, P > 0.05). Similarly, as can be seen in Fig. 6B, saline did not affect the mean response amplitude at threshold (predrug: 89.9 ± 17.22 µV vs. 84.7 ± 10.15 µV after saline, paired t-test, n = 5 pairs, P > 0.05).



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FIG. 6. Muscimol reduced CF stimulus-evoked response amplitude but did not affect threshold or onset latency consistently. A: example of muscimol-induced reduction of response magnitude with no change in threshold (20 dB) or onset latency at most intensities. B: muscimol reduced average peak amplitude of CF stimulus-evoked responses but did not change response threshold (repeated-measures ANOVA, P < 0.001; paired t-test vs. predrug at each intensity, n = 5–9 pairs, P < 0.05, except –10 dB, P » 0.05). Control saline injections had little effect (paired t-test vs. predrug at each intensity, n = 5, P > 0.05). C: muscimol did not alter average onset latency consistently (paired t-test vs. predrug at each intensity, n = 5–9 pairs, P > 0.05 with the exception of 40 dB, P < 0.05).

 
We also analyzed the effect of muscimol on responses to CF stimulus onset. While muscimol appeared to increase onset latency in some cases (e.g., 50 dB response in Fig. 6A), this effect was not consistent. On average, muscimol tended to not alter onset latency (Fig. 6C). The lack of a consistent effect on CF threshold and onset latency suggests that these features depend more on monosynaptic activity—thalamocortical input—than on intracortical processing (see DISCUSSION for full rationale). Conversely, the strong reduction of the response peak amplitude, and the complete reduction of later components of the response, implicates intracortical amplification of thalamocortical input.

The reduction of response magnitude to CF stimuli with no consistent change in onset latency or threshold suggested that muscimol was acting as desired—inhibiting intracortical activity without affecting thalamocortical input. We therefore examined the effect of muscimol on responses to non-CF stimuli because preferential reduction of such responses would support the hypothesis that intracortical pathways mediate non-CF inputs (Fig. 5B), whereas equivalent reduction of responses to CF and non-CF stimuli would suggest that intracortical pathways contribute similarly to both (Fig. 5A). An example of muscimol's effect is shown in Fig. 7A. As in the previous example, muscimol reduced responses to CF stimuli but did not change CF threshold ({uparrow}). However, response amplitudes to non-CF stimuli were also reduced and, in many cases, reduced fully. When averaged data were compared for inhibition of responses to CF versus non-CF stimuli (non-CF data from 1 to 3 octaves below CF combined), only small differences emerged (Fig. 7B). However, the reduction of responses to non-CF stimuli appeared more prominent at the frequencies most spectrally-distant from CF. Because the complete reduction of these responses necessarily would reduce bandwidth, we quantified bandwidth for individual sites and then determined average bandwidth at each intensity. An analysis of bandwidth 20–70 dB above the threshold for CF stimulus-evoked responses showed that muscimol reduced breadth of tuning 20–50 dB above threshold (Fig. 7C; muscimol had no significant effect 60–70 dB above threshold, but note that our detection limit of 5 octaves probably precluded measurement of full predrug bandwidths).



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FIG. 7. Reduction of receptive field bandwidth by muscimol. A: example of effect on tone-evoked responses at mid-frequency site. Intracortical microinjection of muscimol (5.1 mM, 1 µl) reduced response amplitude without affecting CF threshold. Note that responses to some non-CF stimuli appear to be reduced completely. B: response amplitudes in muscimol expressed as a percent of predrug peak amplitude for CF stimuli (n = 5–9 at each intensity) and non-CF stimuli (data from 1 to 3 octaves below CF combined for each experiment, n = 5–9 at each intensity except n = 1 for 10 dB; paired t-test comparing responses evoked by CF stimuli and non-CF stimuli, P < 0.05 at 20 dB). C: muscimol reduced average bandwidth 20–50 dB above CF stimulus-evoked response threshold (paired t-test, n = 5–9 at each intensity, P < 0.05) but not 60–70 dB above threshold (P > 0.05). ···, maximum detectable bandwidth, 5 octaves.

 
We also examined the effects of intracortical muscimol on boosting. Muscimol reduced boosted responses but abolished boosting in only 2/8 animals ("boosting" defined as a response to a non-CF stimulus greater than that to a CF stimulus; premuscimol boosting occurred in 8/9 animals). An example is seen in Fig. 7A, where premuscimol boosting is prominent at 70 dB. Although muscimol strongly reduced all response amplitudes, the response to CF stimuli still appears weaker than the response at –3 octaves (1.25 kHz), i.e., some boosting remained in muscimol (see also group data in Fig. 9A).



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FIG. 9. Picrotoxin diffusing from the recording electrode preferentially disinhibits responses to CF stimuli over responses to non-CF stimuli (3 octaves below CF). A: average data showing greater reversal by picrotoxin of muscimol-inhibited responses to CF stimuli (i) compared with responses to non-CF stimuli (ii; repeated-measures ANOVA, n = 6–8 for each intensity, P < 0.001 for CF and non-CF comparisons: predrug vs. muscimol, muscimol vs. picrotoxin, predrug vs. picrotoxin). B: average responses after muscimol and picrotoxin expressed as percentage of predrug values. Muscimol reduced response amplitude similarly for both CF and non-CF stimuli, but responses to CF stimuli show a greater recovery after picrotoxin (factorial ANOVA comparing CF and non-CF 30–60 dB after picrotoxin, n = 6–8, P < 0.001). Note that the effect of picrotoxin on CF stimulus-evoked responses varied with intensity (regression analysis, R2 = 0.124, P < 0.05; correlation of non-CF stimulus-evoked responses with intensity was not significant).

 
The reduction in mean bandwidth by muscimol suggests that breadth of tuning reflects intracortical processes (Fig. 5B). However, it is also possible that the reduction of responses to near-baseline levels could artifactually produce a reduction in bandwidth by preferentially reducing responses to non-CF stimuli below our response threshold (defined as 2 SDs from the mean baseline, see METHODS). Such an effect on bandwidth might be more prominent at lower intensities, where boosting is not seen and responses to non-CF stimuli are smaller than responses to CF stimuli. To resolve this issue, we designed a final experiment to reverse the muscimol effect preferentially at the recording site and determined the effects of this manipulation on responses to CF and non-CF stimuli.

Local reversal of inhibition at the recording site after inhibition of ACx by muscimol

In this final experiment, we added picrotoxin (a GABAA receptor channel blocker) to the recording electrode after recording responses in muscimol-inhibited ACx (Fig. 8A). Our rationale was the following: if thalamocortical pathways preferentially mediate responses to CF stimuli and intracortical pathways preferentially mediate responses to non-CF stimuli (Fig. 5B), then reversing muscimol's inhibition only at the recording site should preferentially restore responses to CF stimuli. If, on the other hand, thalamocortical inputs contribute equally to CF and non-CF stimulus-evoked responses (Fig. 5A), then reversing muscimol's effect at the recording site should restore responses to CF and non-CF stimuli equally. We therefore determined the effects of local picrotoxin application on intensity functions (LFP amplitude vs. stimulus level) for CF (10 kHz) and non-CF (1.25 kHz) stimuli, using a non-CF stimulus 3 octaves below CF to minimize the possibility that picrotoxin could diffuse to the main cortical representation of the non-CF stimulus.



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FIG. 8. Picrotoxin reverses inihibition by muscimol. A: schematic of experimental design showing picrotoxin diffusing from the electrode to act locally at the recording site, whereas muscimol, previously delivered by intracortical micoinjection, inhibits a much larger region of cortex. B: example showing muscimol (200 µM, 3 µl)-induced reduction of responses to CF and non-CF (–3 octaves) stimuli, and reversal of inhibition by picrotoxin (100 nM in saline). Reversal of inhibition was greater for CF stimulus-evoked responses than for non-CF stimulus-evoked responses.

 
In preliminary experiments, we found that even high concentrations of picrotoxin were ineffective in reversing the effects of 5.1 mM muscimol despite the noncompetitive nature of picrotoxin's block. After experimenting with lower doses of muscimol, we determined that 200 µM muscimol could be antagonized successfully. In eight animals we first recorded intensity functions at CF and non-CF before and after microinjections of muscimol (3–5 µlof200 µM). We then placed an electrode containing picrotoxin (0.01–100 µM) at the recording site and obtained tone-evoked responses, alternating between CF and non-CF stimuli. An example is shown in Fig. 8B, and averaged data are shown in Fig. 9A. The lower concentration of muscimol effectively reduced response amplitudes, and picrotoxin partially reversed the inhibited responses. However, picrotoxin produced greater recovery of responses to CF stimuli than to non-CF stimuli (Figs. 8B and 9). As a control for nonspecific effects, in two animals injection of 3 µl saline at the recording site did not affect responses to CF stimuli (95.6 ± 12.5% of baseline amplitude), whereas 3 µl muscimol reduced amplitudes to 37.8 ± 7.2% of baseline (paired t-test with pooled responses to 40–60 dB stimuli, P < 0.05), and the addition of picrotoxin to the recording electrode reversed the inhibition (87.9 ± 16.3% of control, paired t-test vs. saline, P > 0.05).

The averaged data in Fig. 9Ai show that muscimol markedly reduced response amplitude to CF stimuli and picrotoxin produced partial recovery that approached control amplitudes at high intensities. The average data in Fig. 9Aii show that muscimol also reduced response amplitudes to non-CF stimuli but that reversal by picrotoxin was weaker. In Fig. 9B, plotting the muscimol and picrotoxin data relative to predrug amplitudes reveals more clearly the greater reversal of suppression for response amplitudes to CF versus non-CF stimuli. Note that the picrotoxin effect varied with intensity (CF data in Fig. 9B). These data support the idea that CF information is relayed directly to the recording site by thalamocortical projections, whereas non-CF input arrives preferentially via intracortical pathways (Fig. 5B).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
We have used whole cell intracellular and LFP recordings to investigate how frequency receptive fields within primary ACx are generated. Intracellular recordings revealed that subthreshold receptive fields are substantially broader than suprathreshold (derived from spikes) receptive fields described in this and earlier studies. Receptive fields were equally broad in intracellular and LFP recordings. Cortical microinjection of the GABAA receptor agonist muscimol reduced the bandwidth of LFP-based receptive fields in layer 4, indicating that intracortical pathways contribute to broad receptive fields. Delivery of the GABAA antagonist picrotoxin to the recording site after widespread muscimol-induced inhibition preferentially disinhibited responses to CF stimuli over responses to non-CF stimuli. These data suggest that intracortical integration underlies broad frequency receptive fields in primary ACx.

Intracellular and LFP responses to tones: do LFPs reflect local EPSPs?

In the present study, the principal response features common to whole cell and LFP recordings from the same site included: similar CFs, similar breadth of frequency receptive fields at each intensity tested, receptive field breadth >=5 octaves at moderate to high intensities, and minimum response latencies to CF stimuli and progressively longer onset latencies to stimuli with increasing spectral distance from CF. The main differences between intracellular and LFP responses were that intracellular recordings tended to have higher thresholds at CF and longer latencies than corresponding LFPs. These data indicate that LFPs reflect synaptic potentials in a local group of neurons with similarly broad spectral integration centered on the same CF but with varied thresholds and onset latencies (LFP latencies and thresholds likely reflect those of neurons with the shortest latencies and lowest thresholds).

LFPs are considered an extracellular reflection of synchronous synaptic potentials and are especially clear in laminated structures such as hippocampus and neocortex (Eggermont and Smith 1995Go; Johnston and Wu 1995Go). Data from simultaneous extracellular and intracellular recordings in ACx in vitro and in vivo have demonstrated that electrical stimulation of afferents at intensities that consistently produce subthreshold EPSPs invariably elicits LFPs and synaptic potentials of opposite polarity but with similar latencies (Cruikshank et al. 2002Go; Hsieh et al. 2000Go; Metherate and Ashe 1993bGo, 1994Go; Metherate and Cruikshank 1999Go). In particular, in the auditory thalamocortical slice preparation, MG stimulation evokes layer 4 EPSPs and LFPs with virtually identical onset latencies (Cruikshank et al. 2002Go). Thus LFPs can be useful for exploring synaptic connectivity as in the present study. While the exact volume over which LFP activity can be detected is unknown, note that CFs differed between recording sites separated by <=1 mm. Such differences might not exist if neural activity from distant frequency representations could be detected by the recording electrode and suggest that high-impedance LFP electrodes sample activity over a distance <=1 mm. Although measures of single-unit activity are more precise in terms of localizing neural activity and bear a clearer neural basis, advantages of LFPs include the measurement of presumed subthreshold activity (e.g., can be used to determine breadth of total synaptic input) at predetermined recording sites (e.g., layer 4), and recordings that are reproducible and stable (Barth and Di 1990Go; Eggermont 1996Go; Galvan et al. 2001Go; Norena and Eggermont 2002Go; Ohl et al. 2000Go; Steinschneider et al. 1999Go). Conversely, despite the advantages of LFPs the uncertainty about the volume over which summed neural activity is recorded requires intracellular confirmation of critical features.

Functional connectivity inferred from response to CF stimuli

At a given recording site, CF stimuli evoked the shortest onset latency, which was expected given previous observations of minimum spike latency at CF (Brugge et al. 1969Go; Eggermont 1996Go; Phillips and Hall 1992Go). However, CF stimuli at the high-frequency site evoked shorter latency onsets than did CF stimuli at the mid-frequency site (average: 1.5 ms difference). Shorter minimum spike latencies for higher-frequency CF stimuli have been observed throughout the auditory system, including cochlear nerve (Evans 1972Go; Kiang 1965Go), cochlear nucleus (Kitzes et al. 1978Go), inferior colliculus (Langner et al. 1987Go; Sanes and Constantine-Paton 1985Go), and auditory cortex (Mendelson et al. 1997Go; but see Phillips 1998aGo) and likely result from timing differences in the transduction of acoustic stimuli in the cochlea; i.e., high-frequency stimuli are transduced near the base of the basilar membrane, whereas low-frequency stimuli are transduced further along the basilar membrane closer to its apex. Rough estimates of travel time along the basilar membrane based on published accounts of cochlea, cochlear nerve, and cochlear nucleus recordings (Evans 1972Go; Kiang 1965Go; Kitzes et al. 1978Go; Robles and Ruggero 2001Go) range from 0.3 to 0.7 ms/octave, similar to the ~0.75 ms/octave obtained in the present study. These findings suggest that precise response timing is maintained throughout the auditory system (Heil and Irvine 1997Go). Such precision must reflect specialized synaptic mechanisms at each level of the auditory "labeled line" (Oertel 1999Go; Trussell 1999Go), and similar specializations may exist in ACx as well (Metherate and Aramakis 1999Go).

The notion of a labeled line implies that CF input to neurons in primary ACx comes from MGv neurons with the same CF. Supporting evidence is provided by the effects of muscimol in the present study, which imply long-lasting inhibition of cortical neurons with little effect on afferent (thalamocortical) axons (Edeline et al. 2002Go; Talwar et al. 2001Go). Whereas muscimol significantly reduced LFP amplitude and receptive field bandwidth, CF stimulus-evoked response threshold and onset latency did not change significantly, suggesting that these features rely on monosynaptic thalamocortical afferents (see next section for detailed rationale). Similarly, the lack of a consistent effect on CF stimulus-evoked response threshold and onset latency suggests that muscimol microinjections into the cortex did not diffuse to the thalamus. These findings are consistent with the results of studies using physiological recordings and anatomical tracing to show that like-CF regions of ACx and MG interconnect (Imig and Morel 1984Go; Winer et al. 1999Go). Also, cross-correlated unit recordings of MG and ACx neurons indicate functional connectivity when CFs are within one-third of an octave (Miller et al. 2001Go). Thus thalamocortical afferents convey to cortex information regarding frequencies preferentially at or near CF.

Using muscimol to distinguish thalamocortical from intracortical processes

In theory, inhibition of cortical activity by muscimol does not prevent thalamocortical afferents from releasing transmitter, but the resulting monosynaptic EPSPs would be small amplitude due to sustained shunting of the postsynaptic neuron by open Cl channels and less likely to elicit spikes. The inhibition of postsynaptic spiking would preclude polysynaptic (intracortical) activity. The effect on LFP responses elicited by stimulation of afferents entering the inhibited region would be to reduce amplitudes completely except for the earliest (monosynaptic) portion of the response, which would have an amplitude that was reduced (though not completely) and no change in onset latency. In the present study, muscimol, in the volumes and concentrations used, was expected to diffuse several millimeters from the injection site and produce significant inhibition of neurons over a large portion of primary ACx (Edeline et al. 2002Go; Martin and Ghez 1999Go). CF stimulus-evoked LFPs were reduced as would be expected for preferential inhibition of polysynaptic over monosynaptic activity (i.e., without change in onset latency, with partial reduction of early response components, and with complete suppression of later response components). Moreover, the presence of a presumed monosynaptic LFP component, along with the minimal effects of muscimol on response threshold at CF, suggest that muscimol did not diffuse so far as to reach the MG and inhibit thalamocortical relay neurons. Thus the use of muscimol should help distinguish thalamocortical from intracortical processes.

Because muscimol delivery in the present study involved injection of microliter volumes into ACx, it was necessary to demonstrate that the effects attributed to muscimol did not result from the volume of fluid injected. In addition to the slow rate of injection (0.5–1.0 µl over 1–2 min) and the 15–20 min delay after injection before data collection was begun (to ensure that compression effects dissipated), several results attest to the pharmacological nature of muscimol's actions. First, muscimol reduced neural response amplitude 74%, whereas saline injections of the same volume produced only 7% reductions. Second, in one animal, surface bath application of muscimol produced inhibition similar to that produced by intracortical microinjections in other animals. Third, neither muscimol nor saline significantly affected response threshold—presumably a vulnerable response because of its small amplitude—and saline did not affect response amplitude at threshold (6% reduction). Fourth, the inhibitory effects of muscimol were reversed by picrotoxin after sequential injections of saline and muscimol, whereas damage to the cortex would have resulted in progressively smaller responses after each injection. Together, these results demonstrate that the muscimol's effects were largely due to actions at receptors and not to damage from injected volumes.

In this context, it is not entirely clear why reversal by picrotoxin varied with intensity (Fig. 9B). One possibility is that damage to the recording site from muscimol injections may be more evident at lower intensities, although the lack of significant changes to response threshold after saline or muscimol injections argues against such damage. Alternatively, more intense stimuli may activate greater numbers of neurons and thus may be more likely to include neurons affected by the diffusion of picrotoxin from the recording pipette. In contrast, the few neurons activated at threshold may not include those neurons closest to the recording pipette and the picrotoxin diffusing from it.

Functional connectivity inferred from responses to non-CF stimuli: the basis of broad receptive fields

LFP recordings demonstrated responses to non-CF stimuli <=5 octaves from CF, with onsets occurring progressively later than the onset of responses to CF stimuli. We consider three hypotheses to explain these data, and their implications for functional connectivity in ACx.

NON-CF INPUT IS CARRIED BY THE SAME THALAMOCORTICAL AFFERENTS AS CF INPUT, BUT THALAMIC NEURONS SPIKE AT LONGER LATENCIES TO NON-CF STIMULI THUS PRODUCING LONGER-LATENCY SYNAPTIC ONSETS IN ACX. At all levels of the auditory system, neurons spike to non-CF stimuli at longer latencies than to CF stimuli (Brugge et al. 1969Go; Eggermont 1996Go; Hind et al. 1963Go; Kitzes et al. 1978Go; Phillips and Hall 1992Go). Thus a thalamocortical neuron would relay non-CF information at longer latencies, resulting in longer latency synaptic (and LFP) onsets in ACx. This scenario, while undoubtedly correct, does not seem to account for the onset latencies evoked by non-CF stimuli in the present study. Published quantitative examples of single unit responses to CF and non-CF stimuli indicate approximate changes in minimum spike latency of 12–60 ms/octave for ACx (10–30 dB above threshold, data for intervals <1 octave are extrapolated) (Brugge et al. 1969Go; Eggermont 1996Go; Phillips and Hall 1992Go), 4–8 ms/octave for inferior colliculus (10 dB above threshold) (Hind et al. 1963Go), and 8–15 ms/octave for cochlear nucleus (50–60 dB above threshold) (Kitzes et al. 1978Go). These latency ranges are generally much longer than the 0.7–4.3 ms/octave for 60–10 dB above threshold in the present study (LFP responses in Fig. 3C). Also, the reduction of bandwidth by intracortical muscimol, and the differential disinhibition of responses to CF stimuli over responses to non-CF stimuli by picrotoxin suggests that non-CF information is not carried by the same afferents that relay CF information. Together, these observations indicate that even though thalamic neurons may spike to non-CF stimuli at long latencies, those spikes do not mediate the earliest arrival of non-CF input at the cortical recording site.

NON-CF INPUT IS VIA DIVERGENT THALAMOCORTICAL PROJECTIONS DELIVERING CF INPUT TO OTHER SITES (FIG. 5A). In this scenario, broadly divergent thalamocortical projections provide CF input to one cortical site and non-CF information to other sites. The divergent terminal arbor could produce slightly longer latencies for responses to non-CF stimuli relative to responses to CF stimuli, i.e., the longer path length of thalamocortical collaterals may inc