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Department of Neurobiology and Behavior, University of California, Irvine, California 92697
Submitted 20 November 2003; accepted in final form 20 January 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Merzenich et al. 1975; Phillips et al. 1985b; Sally and Kelly 1988). Similar features are found throughout the lemniscal auditory system (Calford et al. 1983), including in the ventral division of the medial geniculate thalamus (MGv), which provides the main auditory input to primary ACx (Roger and Arnault 1989; Romanski and LeDoux 1993). Because MGv neurons project to cortical neurons with the same CF (Imig and Morel 1984; Miller et al. 2001; Winer et al. 1999), 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. 2001; Muller and Scheich 1988; Wang et al. 2000, 2002). 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 2002; Ribaupierre et al. 1972; Volkov and Galazjuk 1991; Wehr and Zador 2003). 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. 2001; Weinberger and Bakin 1998). 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 1998; Recanzone et al. 1993).
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.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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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 1988). Recording from the cortical surface within this area, we used short-latency, large-amplitude responses to click stimuli to localize ACx (Barth and Di 1990; Shaw 1988) and then made multiple penetrations into layer 4 of the cortex to confirm the tonotopic arrangement characteristic of primary ACx (Doron et al. 2002; Horikawa et al. 1988; Kilgard and Merzenich 1998; Sally and Kelly 1988).
INTRACELLULAR RECORDING. Methods for in vivo whole cell recordings are similar to those described previously (Metherate and Ashe 1993a). 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
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. 1989; Metherate and Ashe 1993a). 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). 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 1993a).
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 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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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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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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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 1993a). 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; 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. 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. After break-in, rhythmic membrane potential fluctuations characterized spontaneous activity as described previously (Metherate and Ashe 1993a; Metherate et al. 1992). 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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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). 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 1990; Metherate and Ashe 1993b; Metherate et al. 1992; Muller-Preuss and Mitzdorf 1984). 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. 2002; Horikawa et al. 1988; Sally and Kelly 1988).
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. 2002; Horikawa et al. 1988; Sally and Kelly 1988). 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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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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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 1999).
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. 2002; Martin and Ghez 1999; present study). Muscimol at such doses would be expected to diffuse several millimeters from the injection site (Edeline et al. 2002; Martin and Ghez 1999) 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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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 (
). 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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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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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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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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 1995; Johnston and Wu 1995). 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. 2002; Hsieh et al. 2000; Metherate and Ashe 1993b, 1994; Metherate and Cruikshank 1999). In particular, in the auditory thalamocortical slice preparation, MG stimulation evokes layer 4 EPSPs and LFPs with virtually identical onset latencies (Cruikshank et al. 2002). 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 1990; Eggermont 1996; Galvan et al. 2001; Norena and Eggermont 2002; Ohl et al. 2000; Steinschneider et al. 1999). 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. 1969; Eggermont 1996; Phillips and Hall 1992). 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 1972; Kiang 1965), cochlear nucleus (Kitzes et al. 1978), inferior colliculus (Langner et al. 1987; Sanes and Constantine-Paton 1985), and auditory cortex (Mendelson et al. 1997; but see Phillips 1998a) 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 1972; Kiang 1965; Kitzes et al. 1978; Robles and Ruggero 2001) 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 1997). Such precision must reflect specialized synaptic mechanisms at each level of the auditory "labeled line" (Oertel 1999; Trussell 1999), and similar specializations may exist in ACx as well (Metherate and Aramakis 1999).
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. 2002; Talwar et al. 2001). 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 1984; Winer et al. 1999). 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. 2001). 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. 2002; Martin and Ghez 1999). 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. 1969; Eggermont 1996; Hind et al. 1963; Kitzes et al. 1978; Phillips and Hall 1992). 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. 1969; Eggermont 1996; Phillips and Hall 1992), 4–8 ms/octave for inferior colliculus (10 dB above threshold) (Hind et al. 1963), and 8–15 ms/octave for cochlear nucleus (50–60 dB above threshold) (Kitzes et al. 1978). 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
