1 Cortical Organization and Systematics, RIKEN BSI, Wako, Saitama 351-0198 and , 2 First Department of Anatomy and , 3 Second Department of Anatomy, Toho University School of Medicine, Toho, Japan
Address correspondence to Hisayuki Ojima, Cortical Organization and Systematics, RIKEN BSI, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Email: yojima{at}brain.riken.go.jp
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Abstract |
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Introduction |
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Studies under anaesthetized or awake conditions have shown that auditory cortical neurons respond distinctly with increased or decreased spike number to different frequencies of sounds (Evans and Whitfield, 1964; Goldstein et al., 1968
, 1970
; Merzenich et al., 1975
; Schreiner and Mendelson, 1990
; Sutter et al., 1999
), differences in sound intensity magnitude or amplitude envelope (Phillips and Irvine, 1981
; Phillips et al., 1985
; Phillips, 1988
; Schreiner et al., 1992
; Heil et al., 1994
; Calford and Semple, 1995
; Sutter and Schreiner, 1995
) and different binaural cues (Hall and Goldstein, 1968
; Brugge et al., 1969
; Brugge and Merzenich, 1973
; Imig and Adrian, 1977
; Middlebrooks et al., 1980
; Middlebrooks and Pettigrew, 1981
; Reale and Kettner, 1986
; Imig et al., 1990
; Semple and Kitzes, 1993a
,b
; Clarey et al., 1994
; Barone et al., 1996
; Reser et al., 2000
).
Several types of suppressions are known to shape the response properties of auditory cortical neurons. These include: (i) lateral suppression, i.e. a reduction in response to a test stimulus by the presentation of a probe stimulus of a different frequency (either prior to or simultaneously with the test stimulus) (Calford and Semple, 1995; Sutter et al., 1999
); (ii) non-monotonic rate versus intensity suppression, i.e. a reduction in response to a test stimulus that results from progressive increases in the intensity of the test signal (Phillips et al., 1985
; Semple and Kitzes, 1993a
,b
; Heil et al., 1994
; Sutter and Schreiner, 1995
); (iii) excitatory/inhibitory binaural interaction suppression, i.e. a reduction in response due to binaural stimulation when compared to the monaural condition (Imig and Brugge, 1978
; Imig and Reale, 1981; Phillips and Irvine, 1983
; Phillips, 1985
; Phillips et al., 1985
; Calford and Semple, 1995
). In order to understand synaptic events underlying these suppressions, in this study we have intracellularly characterized membrane potential responses of pyramidal neurons (PNs) using pure tone stimulation. The results provide evidence for the temporal interactions of depolarization and hyperpolarization underlying the suppressive response properties. Following the physiological characterizations, most neurons were labeled by a tracer injected from the recording pipettes, so that the precise laminar positions of the cell bodies could be determined. In combination with previous anatomical work on local connections, these data contribute to our further understanding of network organization.
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Materials and Methods |
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Animal Preparation
Nineteen healthy cats of both sexes, weighing 2.05.0 kg and having clean external acoustic meati, were pretreated with atropine sulfate (0.025 mg/kg s.c.) and dexamethasone (0.25 mg/kg i.m.) prior to general anesthesia with Nembutal (sodium pentobarbital, 35 mg/kg i.p.; Abbott Laboratories, TX). The cats underwent tracheotomy and cephalic vein catheterization. Anesthesia was maintained by infusion of diluted Nembutal (10 mg/ml saline) through a catheter connected to an infusion pump (CFV-2100; Nihon Kohden, Japan), and delivered continuously at a speed of 0.3 ml/kg/h during recording. Additional volumes of diluted Nembutal were manually administered when necessary. The cats were placed in a stereotaxic apparatus set in a single-walled, sound attenuating chamber (Aco, Japan) and secured to standard ear bars inserted into the external acoustic meati. A post was anchored to the frontal skull with screws and acrylic resin. An antero-posteriorly elongated opening was made in the skull to expose the dura mater. The dura mater was cut above the primary auditory field between the dorsalmost tips of the anterior and posterior ectosylvian sulci or slightly dorsal to this level (Ojima et al., 1991). An acrylic cylindrical chamber (inner diameter 20 mm) was attached to the skull with carbo cement (Shofu, Kyoto, Japan). The ear bars were then removed and a sound delivery system connected to a speaker enclosure (see below) was inserted into each of the external acoustic meati.
The electrocardiogram was continuously monitored, pupil size was observed with a CCD camera and areflexia was periodically checked by pinching a forepaw. The body temperature was maintained at 37.038.0°C using a thermostatically controlled heating pad.
Acoustic Stimuli
Acoustic stimuli were generated digitally by a MalLab system (Kaiser Instruments, Irvine, CA) controlled by a Macintosh computer (He et al., 1997). Acoustic stimulation consisted of pure tone sets, each containing 10 bursts of pure tone of a single frequency (50 ms duration and 10 ms rise/fall time with a sigmoidal envelope, 1.2 s inter-burst interval). After briefly evaluating responses audiovisually at threshold sound intensity, approximately10 sets of tone bursts of different frequencies, from 2 to 25 kHz, were delivered at 10/20 dB above threshold (Sutter et al., 1999
). For intensity functions and binaural interactions, best frequency tones of varied sound intensities with monaural and binaural stimulation (10 repeats for each parameter) were applied. Membrane potentials were monitored together with waveforms of the sound stimuli on an oscilloscope (Kikusui, Japan), and peristimulus time histograms (PSTHs) and raster displays were generated on-line to determine the best frequency (MalLab system). Sound stimulation was delivered through a calibrated silicon tube (2 cm in length and 8 mm in diameter) coupled to an enclosure containing a dynamic earphone speaker (Beyerdynamic DT-48; Beyer, Heilbronn, Germany). The silicon tubes were inserted into the right and left external meati. The gap made between the meatus wall and tube was sealed with Vaseline.
Calibration
The sound delivery system was calibrated at the opening of the silicon tube for a frequency range of 0.125.0 kHz, using a condenser microphone (7017, 0.25 inch; Aco), with reference to a standard sound pressure level (SPL) (94 dB re 20 µPa, 1 kHz, 2126 type; Aco). The calibration data for each ear were stored in a computer file for use in controlling a digital attenuator to obtain desired SPLs.
Recording
Glass microelectrodes (CEI, UK) were pulled on a vertical puller (PE-2; Narishige, Japan) and filled with 1.5% biocytin (Sigma, MO) dissolved in 1.0 M potassium methylsulfate (ICN, OH) or 1.0 M potassium acetate (Sigma). For some neurons, the salt was replaced by 1.0 M potassium chloride (Sigma) buffered with 0.01 M TrisHCl (pH 8.0). Microelectrode resistances ranged from 60 to 80 M. A silver/silver chloride wire and plate were used as a lead from the microelectrode and a reference electrode, respectively. The microelectrode, set on a remote controlled stepping microdrive (PC-5N; Narishige, Japan) was advanced vertically into the brain through the slit in the dura. Brain pulsation was reduced by filling the chamber with warm wax (melting point 45°C). After balancing the null potential level of the recording system, the resistance and capacitance of the microelectrodes were adjusted by bridge balancing and capacity compensation, respectively (MEZ-8301; Nihon Kohden, Japan). Sound stimulation was initiated if the impalement of neurons was followed by an initial shift of membrane potential by more than 40 mV and if injury-induced action potentials disappeared within a few minutes. If these criteria were not met, the microelectrode was advanced further. This through penetration frequently led to degeneration of the neurons. Depth along an electrode track was registered for every neuron impaled. Each hemisphere had between three and five penetration tracks. At most three neurons were recorded per track. When multiple recordings were attempted along one track, the microelectrode was withdrawn by 200 µm towards the cortical surface and then advanced again to a deeper level for the next neuron. This prevented degeneration of the recorded neurons, which would otherwise be caused by the through penetration. To define laminar position accurately, recorded neurons were labeled with biocytin injected iontophoretically (1.52.0 nA positive current for
10 min). Biocytin also diffused spontaneously into neurons while recording and labeled them enough to allow identification of cell morphology if the recording lasted for >5 min. From time of surgery, experiments were completed within 18 h; typically recording sessions lasted <12 h.
Histology
Immediately after finishing recording, animals were administered an overdose of Nembutal and perfused transcardially with saline (300500 ml) followed by 4% paraformaldehyde (Sigma) in 0.1 M phosphate buffer (pH 7.4). The brain was then quickly removed from the skull and kept in the same fixative at 4°C overnight. Visualization of labeled neurons was identical to that described in detail previously (Ojima et al., 1991). Briefly, 50 µm sections were cut on a freezing microtome, reacted in sequence by the ABC method (Vector Laboratories, CA) and 0.06% 3',3'-diaminobenzidine (Sigma). Sections were counterstained with 0.1% thionin (Sigma) and laminar borders were determined on the basis of the sizes and densities of cell bodies (Winguth and Winer, 1986
). Layer 2 was 150200 µm thick, corresponding to between five and six rows of cell bodies (Fig. 1
). Reconstruction of microelectrode tracks and positions of labeled cell bodies were used to identify the recording points on labeled neurons.
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Recording of single PNs typically lasted 1090 min. Membrane potentials, sound stimuli, current injected and trigger signals were recorded on a 4-channel digital audio tape recorder (DC to 10 kHz, PC204Ax; Sony, Japan) together with voice commentary. Parameters were analyzed off-line on a PowerLab system (AD Instruments, Australia).
Recordings were made from a total of 193 cortical PNs. For detailed off-line analysis, the following selection criteria were used: stable resting membrane potentials greater than 50 mV, responses to a wide range of pure tone bursts sufficient to define the best frequencies, identified laminar position of the cell bodies and a recording point close to the cell body (<50 µm). Forty-seven PNs fulfilled the above criteria. Their resting membrane potentials ranged from 50.1 to 70.3 mV. An additional seven PNs could not be driven by or tuned to pure tones. The extent of diffusion of the tracer in these PNs varied from light labeling of the cell body/proximal dendrites to intense labeling of both cell body/dendrites and axons (Fig. 1).
In some traces of membrane potential, spontaneous action potentials or depolarizations comparable in amplitude to acoustically induced ones occurred during a 50 ms pre-stimulus period. These traces were excluded from the data analysis, since activation prior to the stimulus is known to affect stimulus-induced responsiveness (Ferster and Jagadeesh, 1992; Ganguly et al., 2000
; Henze and Buzsáki, 2001
). The remaining traces within each trial were averaged to obtain a mean trace of the membrane potentials. In some cases, for comparison of the peak amplitudes of membrane potentials induced by different stimulation variables, each trace was low-pass filtered at 100200 Hz to eliminate action potentials and then subjected to averaging.
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Results |
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Membrane potential changes of layer 2 and 3 PNs were examined in response to a variety of pure tone bursts applied binaurally at the resting membrane potential level. There were at least three common characteristics in the membrane potential response of PN populations in layers 2 and 3.
Onset depolarization (O-DEP) was induced over a frequency range which was wider than that for action potential generation. Although the response frequency range was wide, a single best frequency could be determined for most PNs on the basis of the O-DEP amplitude (see larger symbols in Fig. 2). In relation to the frequencylatency function for O-DEP, minima were almost always observed (43/44) at the best frequency (Fig. 2
). Often the frequencylatency function had a second minimum at frequencies other than the best frequency (see for example filled square in layer 2 and open diamond in layer 3d in Fig. 2
), consistent with recent extracellular studies (Loftus and Sutter, 2001
). The intensitylatency relationship showed that as sound intensity of a pure tone increased, the latency of O-DEP concomitantly became shorter, reaching a minimum value at the highest intensity (see for example Brugge et al., 1969
; Phillips and Irvine, 1981
). This relationship was observed regardless of whether the O-DEP amplitude increased (monotonic pattern) or decreased (non-monotonic pattern) with increasing sound intensity. The observed latency relationships with frequency and intensity were similar in layer 2 and 3 PNs.
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Frequency Tuning
O-DEP induced in layer 2 PNs was not followed by hyperpolarization. Rather, the O-DEP was followed by a sustained, late depolarization. The amplitude and duration of the late depolarization varied considerably from neuron to neuron. The overall duration of the onset and sustained parts of the depolarization was, on average, 181.3 ± 61.6 ms (n = 6) at the best frequency at 10/20 dB above threshold.
In Figure 5 we show representative membrane potential responses to sets of pure tone bursts at a fixed sound intensity (50 dB) for a layer 2 PN. A frequency range from
6 to 16 kHz was effective in inducing various amplitudes of O-DEP (Fig. 5A
). The averaged O-DEP amplitude (measured from resting membrane potential level) was maximal at a frequency of 14.3 kHz (Fig. 5B,C
). The O-DEP induced at this frequency occasionally led to initiation of action potentials. This frequency corresponded to the best frequency defined by spike counting (Fig. 5D
). Onset latency of the O-DEP was shortest at the best frequency (Fig. 5C,D
).
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(i) Response level function suppression of sound intensity.
Amplitude of O-DEP either monotonically increased with increasing loudness or increased and then decreased as sound intensity increased. Three of six layer 2 PNs examined displayed a suppressive response level relationship of O-DEP (Fig. 6A). In this study, PNs were regarded as non-monotonic if the amplitude of O-DEP was suppressed to
70% of the maximal value. Figure 6B
shows individual traces of membrane potential of a representative layer 2 PN in response to varied sound intensities. As sound intensity increased, the O-DEP amplitude started to increase at the threshold intensity of 40 dB SPL, reached its maximal value at 50 dB SPL and then declined to 67% of the maximum at 70 dB SPL. As a consequence of the relationship between O-DEP latency and sound intensity, the latency of the O-DEP was shortest at the highest sound intensity (upper panel in Fig. 6B
). This O-DEP was not followed by hyperpolarization at any magnitude of sound intensity. Even when the membrane potential was depolarized by passing a positive current (+0.2 nA) through the recording pipette, there was no hyperpolarization following the O-DEP (filled symbols in upper panel and uppermost traces in lower panel of Fig. 6B
). Another example is shown in Figure 6C
, in which membrane potentials are averaged for different SPLs.
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Frequency Tuning
As in layer 2 PNs, the frequency range over which O-DEP was induced in layer 3 PNs was wider than the frequency range over which action potentials were induced (Figs 2 and 8). In Figure 8
we show individual traces of membrane potential (Fig. 8A
) in response to tone frequencies ranging from 5.1 to 11.0 kHz at 30 dB SPL (10 dB above threshold), spike rates (Fig. 8B
) at the best (7.5 kHz) and nearby (8.0 kHz) frequencies and the frequency latency relationship (Fig. 8C
). Hyperpolarization followed large amplitude O-DEP in response to tone bursts whose frequency ranged from 6.1 to 11.0 kHz (Fig. 8A
for individual traces and Fig. 8D
for averaged traces). The largest amplitude hyperpolarization was elicited at the best frequency (i.e. 7.37.5 kHz; Fig. 8D
). At frequencies flanking the best frequency (i.e. 5.1, 6.1 and 11.0 kHz), large amplitude O-DEPs were also elicited, but no action potentials were initiated from the O-DEP (subthreshold depolarization). Following the subthreshold O-DEP, hyperpolarization was also elicited, with an amplitude smaller than that induced at the best frequency (Fig. 8D
). The sequence of subthreshold depolarization and subsequent hyperpolarization at frequencies other than the best frequency occurred for 56% of layer 3 PNs (18 of 32 layer 3 PNs that had acoustically induced hyperpolarization).
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Thirty-five layer 3 PNs were tested for response level changes at their best frequencies. Non-monotonic response level suppression in membrane potential behavior was observed in 63% of the PNs (22/35). Twenty-eight layer 3 PNs were tested for binaural interactions. Suppressive binaural interaction (EI type) was found in 21% of the PNs (6/28).
(i) Response level function suppression of sound intensity.
Of the population of layer 3 PNs which showed a non-monotonic relationship between sound intensity and depolarization amplitude, nearly half (12/22) showed a strong decrease in O-DEP amplitude (70% of the maximal value) with increasing sound intensities. The rest displayed only a weak non-monotonic response of membrane potential.
For the strongly non-monotonic population, the decreased O-DEP amplitude was associated with a shortened duration (Fig. 9A). The shortening of the O-DEP duration resulted mainly from the earlier onset of hyperpolarization following the O-DEP at higher sound intensities. Membrane potentials of a layer 3 PN with a representative non-monotonic response level relationship are shown in Figure 9B
. As sound intensity increased from threshold (35 dB SPL), the amplitude of O-DEP increased and reached a maximal level at 45 dB SPL. The O-DEP amplitude then declined to lower levels at increasingly higher sound intensities (upper panel in Fig. 9B
). In association with the decline in the O-DEP amplitude, the O-DEP duration became shortened (lower panel of Fig. 9B
). In this cell, the O-DEP latency changed from 16.9 to 12.4 ms with a SPL change from 45 to 70 dB (4.5 ms shortening; arrowheads in Fig. 9B
). The latency change of the hyperpolarization was greater in magnitude and ranged from 42.2 to 29.5 ms (12.7 ms shortening; short arrows in Fig. 9B
). This greater degree of shortening of the hyperpolarization latency resulted in shortening of the O-DEP duration. Another example showing a similar non-monotonic relationship is shown in Figure 9C
.
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(ii) Binaural interaction suppression.
A binaural suppression response of membrane potential was also observed. Monaural stimulation of a dominant ear evoked a larger amplitude O-DEP than did binaural stimulation of both ears. Although the number of PNs displaying the binaural suppression interaction was small (six of 28 neurons tested), there were at least three variations in the pattern of membrane potential response (Fig. 10A).
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The second pattern of suppression in the binaural interaction was found in two PNs (gray filled symbols, Fig. 10A). This suppression was not mediated by the strong hyperpolarization generated by non-dominant ear stimulation, but had a complicated temporal interaction of depolarization and hyperpolarization. An example (gray filled diamond, Fig. 10A
) is illustrated in Figure 10C
. This PN is of EI type with an ipsilateral (IPSI) ear dominance, as defined both by spike counting (upper panel) and the amplitude of O-DEP at relatively high sound intensities. As shown in the lower panel, the dominant O-DEP induced ipsilaterally was later relative to the non-dominant O-DEP induced contralaterally. This temporal sequence led to concurrence of the peak of the dominant (IPSI) depolarization with the falling phase of the preceding, non-dominant (CONTRA) depolarization. Although the ear dominance is opposite, the other neuron (gray filled triangle, Fig. 10A
) showed the same temporal sequence of dominant and non-dominant ear responses (compare the latencies of largest and smallest filled symbols in Fig. 10A
).
The other three PNs (open symbols, Fig. 10A) showed the third pattern of membrane potential response underlying the binaural suppression of spike rate. A representative example (open triangle, Fig. 10A
) is shown in Figure 10D
. Dominant CONTRA ear stimulation induced the highest spike rate (0.43 spikes/trace) and largest O-DEP amplitude. Unlike the second pattern, the dominant (CONTRA) depolarization preceded the non-dominant (IPSI) depolarization. This temporal sequence did not lead to concurrence of the peak of the dominant (CONTRA) depolarization with the falling phase of the non-dominant (IPSI) depolarization.
Membrane potential responses associated with EE (excitatory/excitatory) and EO (excitatory/null) type interactions were also observed in layer 3 PNs. Their membrane potential responses were similar to those of the third EI pattern in terms of the temporal relationship, although the binaural O-DEP amplitude was larger than or equal to the dominant, monaural O-DEP amplitude.
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Discussion |
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Activation of Excitatory and Inhibitory Postsynaptic Potentials by Sound Stimulation
This study shows that PNs in the supragranular layer of cat primary auditory cortex respond with O-DEP with or without subsequent hyperpolarization to pure tone bursts in a population-dependent manner. It is likely that the depolarization and hyperpolarization induced by pure tone stimuli reflect excitatory and inhibitory postsynaptic potentials. This interpretation is supported by the following four points. (i) Early hyperpolarizing deflection from the resting membrane potential is believed to reflect an inhibitory postsynaptic potential if the resting membrane potential is set higher (i.e. depolarized) than the equilibrium potential for chloride ions (around 75 mV) (see McCormick, 1998). Indeed, the resting membrane potentials of the PNs examined were between 50 and 70 mV (mostly 50 to 60 mV). (ii) Inhibition is also induced by a shunting mechanism, by which local excitation can be suppressed by an increase in membrane conductance. In this situation, hyperpolarization cannot be manifested if the membrane potential is close to the equilibrium potential for chloride ions. Our trials, in which the membrane potential was set at more depolarized levels, still did not reveal hyperpolarization in layer 2 PNs. It is thus unlikely that the shunting mechanism explains the layer 2 PN suppression observed in cat primary auditory cortex. In other cortical areas shunting inhibition is known not to operate or to do so only weakly (Douglas et al., 1988
; Berman et al., 1991
; Ferster and Jagadeesh, 1992
; however, see Borg-Graham et al., 1998
). (iii) Depolarizing and hyperpolarizing shifts of the base membrane potential by constant current application also leads to increased and decreased amplitude of the hyperpolarization following the O-DEP. Since the amplitude of the hyperpolarization increases after the resting membrane potential is shifted to depolarized levels, this hyperpolarization should not be a manifestation of the transient reduction in excitatory depolarization (for more details see Berman et al., 1991
). (iv) In addition, in three layer 3 PNs, chloride ions were iontophoretically injected through a recording pipette containing potassium chloride in order to shift the equilibrium potential for chloride ions toward a more positive (i.e. depolarized) value. Injection resulted in a diminished amplitude of the hyperpolarization following O-DEP (data not shown; see Krnjevi
and Schwartz, 1967
). Together, these results strongly suggest that the hyperpolarization is a chloride ion-mediated inhibitory postsynaptic potential. Furthermore, as in the visual system, it is also likely that the in vivo inhibitory postsynaptic potential following the preceding excitatory postsynaptic potential induced by acoustic stimulation is mediated by GABAA receptors (Conners et al., 1988
).
Differences in the Membrane Potential Responses of Layer 2 and 3 PNs
Intracellular recording in the present study has characterized excitatory and inhibitory postsynaptic potentials induced in PNs in layers 2 and 3. Although the absolute duration and magnitude of these membrane potentials varied considerably from neuron to neuron, there was a consistent difference in the membrane potential responses of PNs located in these two layers, as determined by dye injection.
In layer 3, most PNs displayed a sequence of excitation and hyperpolarizing inhibition. In contrast, virtually all layer 2 PNs displayed an onset depolarization which is not followed by a hyperpolarization, but rather by a sustained late depolarization. Could the different membrane potential responses be due to a different equilibrium potential for chloride ions of the two PN populations? That is, the chloride equilibrium potential level might be higher (i.e. more depolarized) for layer 2 PNs than their resting membrane potential level. This possibility can be excluded because we were able to confirm the failure in the hyperpolarization induction in layer 2 PNs by setting the membrane potential at a more depolarized level (Avoli, 1986; Ferster, 1986
; Avoli and Olivier, 1989
) (see Fig. 6B
). This membrane potential level also excludes the possibility of involvement of a shunting inhibition mechanism (see above). Also to be considered is whether the stimulus intensity might not be strong enough to induce the hyperpolarization following O-DEP. This possibility seems unlikely, since the hyperpolarization was not induced even when the stimulus intensity was raised to the maximal value (80 dB SPL; Fig. 6C
), which consistently elicited large magnitudes of the hyperpolarization in layer 3 PNs.
Although relevant data are not available for the auditory system, a similar membrane potential behavior has been reported for PNs in in vitro cat motor cortex. Electrical stimulation of superficial layer 3 elicited a large amplitude hyperpolarization in layer 2 PNs, whereas that of deep layer 3 evoked no hyperpolarization (Kang et al., 1994). This raises the possibility that polysynaptic excitation may result in a depolarization without a following hyperpolarization. Anatomical findings have shown that the medial geniculate-cortical afferent projection terminates mainly in layers 3 and 4 (Hashikawa et al., 1995
; Smith and Populin, 2001
) and that PNs in the lower half of layer 3 (and also in layer 4) are the major population activated monosynaptically by thalamic afferents (Smith and Populin, 2001
). Thus layer 2 PNs may not be monosynaptic targets of the thalamic afferent (for the visulal system see Ferster and Lindstrom, 1983
). Since other anatomical findings in both visual (Gilbert and Wiesel, 1979
; Martin and Whitteridge, 1984
) and auditory cortices (Ojima et al., 1991
) have shown that a rich axon plexus extends from layer 3 PNs into layer 2, it is likely that a tonally driven excitation mediated by the thalamic axons can reach layer 2 neurons polysynaptically via excitatory synapses of the layer 3 PNs (Mitani and Shimokouchi, 1985
). This polysynaptic excitatory connection may not activate inhibitory inputs, via interneurons, to layer 2 PNs in the present stimulus paradigm.
ResponseIntensity Relationships
The present findings show that onset time of a sequence of depolarization and hyperpolarization varies depending on tonal frequency, intensity and ear of stimulation (i.e. monaural or binaural). Membrane potential responses of layer 2/3 PNs display a non-monotonic as well as monotonic pattern to increasing SPLs.
One explanation of the decreased amplitude of depolarization in non-monotonic layer 3 PNs follows from the fact that depolarization and hyperpolarization counteract each other in a summative fashion when they temporally coincide (Eccles, 1964, 1968
). Our results show that at higher sound intensities the latency of the hyperpolarization following depolarization becomes shorter. Because of this earlier onset of hyperpolarization, it is likely that the counteraction between the preceding depolarization and subsequent hyperpolarization begins earlier, leading to a shortened duration of the depolarization (Fig. 9A and B
). This shortened duration can result in a decrease in the peak amplitude of the depolarization for nonmonotonic PNs.
The O-DEP induced in layer 2 PNs is not followed by hyperpolarization. Because of the lack of hyperpolarization, it is unlikely that inhibitory inputs to layer 2 PNs are engaged in reducing the amplitude of their O-DEPs. There are at least two explanations for the decreased magnitude of the O-DEP in non-monotonic layer 2 PNs. First, the decreased amplitude of the O-DEP in non-monotonic layer 2 PNs can be thought to result from the decreased number of excitatory inputs activated at higher sound intensities. This situation is equivalent to a situation where stimulation is given at lower sound intensities. Lower sound intensities should induce lower O-DEP amplitudes but result in longer onset latencies of O-DEP. However, this is not the case, since the onset latency of the O-DEP of non-monotonic layer 2 PNs gradually shortens as sound intensity increases (Fig. 3). Second, there may be a preference for layer 2 PNs to receive afferents from layer 3 PNs with similar response features (nonmonotonic). As discussed above, layer 2 PNs are likely to receive excitatory afferents from layer 3 PNs (Ojima et al., 1991
; Kang et al., 1994
) and these connections can be activated by the present stimulation paradigm. If this layer 3 PN population responds in a non-monotonic manner and converges on subsets of layer 2 PNs, the depolarization of these layer 2 subsets are also likely to have a non-monotonic response pattern. In this case, the hyperpolarization cannot necessarily be generated after the depolarization. Furthermore, since the O-DEP latency shortens in non-monotonic layer 3 PNs with increasing sound intensity, this temporal shift of the O-DEP latency should also be reflected in the temporal pattern of O-DEP of layer 2 PNs. This is indeed the case (Figs 3 and 6B
). It can be hypothesized that non-monotonic layer 3 PNs are preferentially connected to non-monotonic layer 2 PNs.
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In layer 3 PNs of EI type, ipsilaterally and contralaterally induced synaptic potentials interact with each other to suppress the magnitude of the binaurally induced membrane potential. From a sample of six neurons, three patterns of suppressive interaction in membrane potential response are suggested.
The first and second patterns of binaural suppression can be explained by the temporal coincidence of depolarization and hyperpolarization induced by dominant and non-dominant ear stimulation. In the first pattern (see Fig. 10B), stimulation of the non-dominant, inhibitory ear alone induces a strong hyperpolarization inhibition with no preceding depolarization. This inhibitory response induced by non-dominant ear stimulation is likely to be responsible for the binaural suppression. Our findings show that the hyperpolarization initiated by the inhibitory ear stimulation temporally coincides with the depolarization peak induced by the excitatory ear stimulation. This enables the hyperpolarization to counteract the depolarization induced by the dominant ear stimulation to suppress its peak/falling phase. In the second pattern of binaural suppression (see Fig. 10C
), the non-dominant ear does not induce a strong hyperpolarization inhibition, unlike the first pattern. Dominant ear stimulation alone rather induces a depolarization and hyperpolarization sequence. The onset/peak of the depolarization induced by dominant ear stimulation is delayed relative to that of the depolarization induced by non-dominant ear stimulation alone. Because of this delay, the dominant depolarization can coincide temporally with the hyperpolarization inhibition following the non-dominant depolarization (Calford and Semple, 1995
). On binaural stimulation, this temporal interaction can result in a partial or full suppression of the dominant depolarization component, leading to a smaller amplitude depolarization on binaural stimulation than on dominant ear stimulation.
In the third pattern of binaural suppression (Fig. 10D), the O-DEP peak induced by dominant ear stimulation does not coincide with the hyperpolarization inhibition following the O-DEP induced by non-dominant ear stimulation. So the reduced magnitude of the depolarization induced binaurally cannot be explained by temporal summation of the membrane potentials. Such a response pattern would result from convergence of presynaptic neurons, located in binaural bands of the same features in the cortex (Imig and Adrian, 1977
; Imig and Brugge, 1978
; Middlebrooks et al., 1980
) and/or medial geniculate body (Calford and Webster, 1981
; Middlebrooks and Zook, 1983
; Rodrigeus-Dagaeff et al., 1989
).
Subthreshold Responses and their Relationships to Lateral Inhibition
The present study shows that layer 3 PNs respond to pure tone bursts with a sequence of depolarization and hyperpolarization at their best and flanking frequencies. This temporal sequence can underlie a phenomenon named forward masking inhibition that has been described in primary auditory cortex (Calford and Semple, 1995). In forward masking, two pure tone stimuli are applied sequentially. A first pure tone (masker tone) with varied frequencies precedes a second pure tone (probe tone) with a fixed frequency (usually the best frequency). It is known that following masker tone stimulation at the best frequency, spike rate on probe tone stimulation is reduced. Such suppression can be explained by a temporal interaction of the hyperpolarization following the masker tone depolarization with the subsequent probe tone depolarization, i.e. if the probe tone depolarization coincides temporally with the hyperpolarizing inhibition that follows the preceding masker tone depolarization, the probe tone depolarization can be suppressed.
A spike rate suppression effect by tones with frequencies outside the response field of a neuron (lateral suppression) can also be explained by a similar temporal interaction. The occurrence of a sequence of subthreshold depolarization and following hyperpolarization suggests that probe tone depolarization can be suppressed if the masker tone stimulation induces a sequence of subthreshold depolarization followed by hyperpolarizing inhibition at frequencies flanking the best frequency (see Fig. 8A and D).
Relationship to Two Tone Simultaneous Masking
Cortical suppression has also been observed when the masker and probe tones are presented simultaneously (Sutter et al., 1999). The probe tone has a fixed intensity (10/20 dB above threshold) at the best frequency and the masker tone has various combinations of frequency and intensity. Suppression of the probe tone responses by the masker tone is observed when the masker tone is applied at frequencies neighboring the best frequency and at higher intensities.
Some of the response properties in this simultaneous suppression (see fig. 11 in Sutter et al., 1999) may be explained by latency dissociation of the masker and probe tone responses. The present data show that the onset latency of depolarization is shorter at the best frequency than at any other frequencies (see Fig. 2
). They also show that the O-DEP latency becomes shorter as sound intensity increases (see Fig. 3
). These results indicate that, depending on the combinations of frequency and intensity, the onset latency of the masker tone depolarization can become shorter relative to that of the probe tone depolarization. Because of this dissociation in latency, it is possible that the probe tone depolarization can coincide temporally with the hyperpolarizing inhibition following the masker tone depolarization.
Conclusions
Using intracellular recording from primary auditory cortical PNs, we have demonstrated membrane potential responses that can explain forward masking, lateral inhibition, non-monotonic rate level suppression and binaural suppression. These suppressive responses can be determined by the temporal interactions of excitatory and inhibitory membrane potentials.
Another mechanism underlying the suppressive responses could also be suggested. Membrane potential responses of a postsynaptic neuron may reflect the convergence of similarly responding presynaptic neurons. The suppressive membrane potential responses of layer 2 PNs and some binaural suppression in layer 3 PNs are likely to be explained by this mechanism.
The present results suggest that PNs in primary auditory cortex act as band-pass filters whose specific characteristics are defined by the temporal interactions of frequency-, level- and monaural/binaural-dependent excitation and inhibition. These interactions also show a laminar-specific distinction.
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Avoli M, Olivier A (1989) Electrophysiological properties and synaptic responses in the deep layers of the human epileptogenic neocortex in vitro. J Neurophysiol 61:589606.
Barone P, Clarey JC, Irons WA, Imig TJ (1996) Cortical synthesis of azimuth-sensitive single-unit responses with nonmonotonic level tuning: a thalamocortical comparison in the cat. J Neurophysiol 75:12061220.
Berman NJ, Douglas RJ, Martin KAV, Whitteridge D (1991) Mechanisms of inhibition in cat visual cortex. J Physiol 440:697722.[Abstract]
Berman NJ, Douglas RJ, Martin KAV (1992) GABA-mediated inhibition in the neuronal networks of visual cortex. Prog Brain Res 90:443476.[ISI][Medline]
Borg-Graham LJ, Monier C, Fregnac Y (1998) Visual input evokes transient and strong shunting inhibition in visual cortical neurons. Nature 393:369373.[ISI][Medline]
Brugge JF, Merzenich MM (1973) Responses of neurons in auditory cortex of the macaque monkey to monaural and binaural stimulation. J Neurophysiol 36:11381159.
Brugge JF, Dubrovsky NA, Aitkin LM, Anderson DJ (1969) Sensitivity of single neurons in auditory cortex of cat to binaural tonal stimulation; effects of varying interaural time and intensity. J Neurophysiol 32:10051024.
Calford MB, Semple MN (1995) Monaural inhibition in cat auditory cortex. J Neurophysiol 73 18761981.
Calford MB, Webster WR (1981) Auditory representation within principal division of cat medial geniculate body: an electrophysiological study. J Neurophysiol 45:10131028.
Casseday JH, Ehrlich D, Covey E (1994) Neural tuning for sound duration: role of inhibitory mechanisms in the inferior colliculus. Science 264:847850.[ISI][Medline]
Clarey JC, Barone P, Imig TJ (1994) Functional organization of sound direction and sound pressure level in primary auditory cortex of the cat. J Neurophysiol 72: 23832405.
Code RA, Winer JA (1986) Columnar organization and reciprocity of commissural connections in cat primary auditory cortex (AI). Hear Res 23:205222.[ISI][Medline]
Conners BW, Malenka RC, Silva LR (1988) Two inhibitory postsynaptic potentials, and GABAA and GABAB receptor-mediated responses in neocortex of rat and cat. J Physiol 406:443468.[Abstract]
Covey E, Kauer JA, Casseday JH (1996) Whole-cell patch-clamp recording reveals subthreshold sound-evoked postsynatpic currents in the inferior colliculus of awake bats. J Neurosci 16:30093018.
de Ribaupierre F, Goldstein MH Jr, Yeni-Komshian G (1972) Intracellular study of the cats primary auditory cortex. Brain Res 48:185204.[ISI][Medline]
Douglas RJ, Martin KAC (1991) A functional microcircuit for cat visual cortex. J Physiol 440:735769.[Abstract]
Douglas RJ, Martin KAC, Whitteridge D (1988) Selective responses of visual cortical cells do not depend on shunting inhibition. Nature 332:642644.[ISI][Medline]
Douglas RJ, Martin KAC, Whitteridge D (1991) An intracellular analysis of the visual responses of neurones in cat visual cortex. J Physiol 440:659696.[Abstract]
Eccles JC (1964) The physiology of synapses. Berlin: Springer-Verlag.
Eccles JC (1968) The physiology of nerve cells. Baltimore: Johns Hopkins University Press.
Evans EF, Whitfield IC (1964) Classification of unit responses in the auditory cortex of the unanaesthetized and unrestrained cat. J Physiol 171:476493.[ISI]
Ferster D (1986) Orientation selectivity of synaptic potentials in neurons of cat primary visual cortex. J Neurosci 6:12841301.[Abstract]
Ferster D (1992) The synaptic inputs to simple cells of the cat visual cortex. Prog Brain Res 50:423441.
Ferster D, Jagadeesh B (1992) EPSP-IPSP interactions in cat visual cortex studied with in vivo whole-cell patch recording. J Neurosci 12:12621274.[Abstract]
Ferster D, Lindstrom S (1983) An intracellular analysis of geniculo-cortical connectivity in area 17 of the cat. J Physiol 342:181215.[Abstract]
Ferster D, Miller KD (2000) Neural mechanisms of orientation selectivity in the visual cortex. Annu Rev Neurosci 23:441471.[ISI][Medline]
Ganguly K, Kiss L, Poo M-M (2000) Enhancement of presynaptic neuronal excitability by correlated presynaptic and postsynaptic spiking. Nature Neurosci 3:10181026.[ISI][Medline]
Gilbert CD, Wiesel TN (1979) Morphology and intracortical projections of functionally characterised neurones in the cat visual cortex. Nature 280:120125.[ISI][Medline]
Goldstein MH Jr, Hall JL, Butterfield BO (1968) Single-unit activity in the primary auditory cortex of unanesthetized cats. J Acoust Soc Am 43:444455.[ISI][Medline]
Goldstein MH, Abeles M, Daly RL, McIntosh J (1970) Functional architecture in cat primary auditory cortex: tonotopic organization. J Neurophysiol 33:188197.
Hall JL, Goldstein MH (1968) Representation of binaural stimuli by single units in primary auditory cortex of unanesthetized cat. J Acoust Soc Am 43:456461.[ISI][Medline]
Hashikawa T, Molinari M, Rausell E, Jones EG (1995) Patchy and laminar terminations of medial geniculate axons in monkey auditory cortex. J Comp Neurol 362:195208.[ISI][Medline]
He J, Hashikawa T, and Ojima H, Kinouchi Y (1997) Temporal integration and duration tuning in the dorsal zone of cat auditory cortex. J Neurosci 17:26152625.
Heil P, Rajan R, Irvine DR (1994) Topographic representation of tone intensity along the isofrequency axis of cat primary auditory cortex. Hear Res 76:188202.[ISI][Medline]
Henze DA, Buzsáki G (2001) Action potential threshold of hippocampal pyramidal cells in vivo is increased by recent spiking activity. Neuroscience 105:121130.[ISI][Medline]
Huang CL, Winer JA (2000) Auditory thalamocortical projections in the cat: laminar and areal patterns of input. J Comp Neurol 427:302331.[ISI][Medline]
Imig TJ, Adrian HO (1977) Binaural columns in the primary field (A1) of cat auditory cortex. Brain Res 138:241257.[ISI][Medline]
Imig TJ, Brugge JF (1978) Sources and terminations of callosal axons related to binaural and frequency maps in primary auditory cortex of the cat. J Comp Neurol. 182:637660.[ISI][Medline]
Imig TJ, Reale RA (1980) Patterns of cortico-cortical connections related to tonotopic maps in cat auditory cortex. J Comp Neurol 192: 293332.[ISI][Medline]
Imig TJ, Irons WA, Samson FR (1990) Single-unit selectivity to azimuthal direction and sound pressure level of noise bursts in cat high-frequency primary auditory cortex. J Neurophysiol 63:14481466.
Kang Y, Kaneko T, Ohishi H, Endo K, Araki T (1994) Spatiotemporally differential inhibition of pyramidal cells in the cat mortor cortex. J Neurophysiol 71: 280293.
Krnjevi K, Schwartz S (1967) The action of
-aminobutyric acid on cortical neurones. Exp Brain Res 3:320336.[ISI][Medline]
Kuwada S, Batra R, Yin TC, Oliver DL, Haberly LB, Stanford TR (1997) Intracellular recordings in response to monaural and binaural stimulation of neurons in the inferior colliculus of the cat. J Neurosci 17:75657581.
Loftus WC, Sutter ML (2001) Spectrotemporal organization of excitatory and inhibitory receptive fields of cat posterior auditory field neurons. J Neurophysiol 86:475491.
Martin KA, Whitteridge D (1984) Form, function and intracortical projections of spiny neurones in the striate visual cortex of the cat. J Physiol 353:463504.[Abstract]
McCormick DA (1998) Membrane properties and neurotransmitter actions. In: The synaptic organization of the brain (Shepherd GM, ed), pp. 3775. New York: Oxford University Press.
Merzenich MM, Knight PL, Roth GL (1975) Representation of the cochlea within primary auditory cortex in the cat. J Neurophysiol 38: 231249.
Middlebrooks JC, Pettigrew JD (1981) Functional classes of neruons in primary auditory cortex of the cat distinguished by sensitivity to sound location. J Neurosci 1:107120.[ISI][Medline]
Middlebrooks JC, Zook JM (1983) Intrinsic organization of the cats medial geniculate body identified by projections to binaural response-specific bands in the primary auditory cortex. J Neurosci. 3:203224.[Abstract]
Middlebrooks JC, Dykes RW, Merzenich MM (1980) Binaural response-specific bands in primary auditory cortex (AI) of the cat: topographical organization orthogonal to isofrequency contours. Brain Res 181:3148.[ISI][Medline]
Mitani A, Shimokouchi M (1985) Neuronal connections in the primary auditory cortex: an electrophysiological study in the cat. J Comp Neurol. 235:417429.[ISI][Medline]
Mitani A, Shimokouchi M, Itoh K, Nimura S, Kudo M, Mizuno N (1985) Morphology and laminar organization of electrophysiologically identified neurons in the primary auditory cortex in the cat. J Comp Neurol 235:430447.[ISI][Medline]
Ojima H, Honda CN, Jones EG (1991) Patterns of axon collateralization of identified supragranular pyramidal neurons in the cat auditory cortex. Cereb Cortex 1:8094.[Abstract]
Ojima H, Honda CN, Jones EG (1992) Characteristics of intracellularly injected infragranular pyramidal neurons in cat primary auditory cortex. Cereb Cortex 2:197216.[Abstract]
Pedemonte M, Torterolo P, Velluti RA (1997) In vivo intracellular characteristics of inferior colliculus neurons in guinea pigs. Brain Res 759: 2431.[ISI][Medline]
Phillips DP (1985) Temporal response features of cat auditory cortex neurons contributing to sensitivity to tones delivered in the presence of continuous noise. Hear Res 19:253268.[ISI][Medline]
Phillips DP (1988) Effect of tone-pulse rise time on rate-level functions of cat auditory cortex neurons: excitatory and inhibitory processes shaping responses to tone onset. J Neurophysiol 59:15241539.
Phillips DP, Irvine DRF (1981) Responses of single neurons in physiologically defined primary auditory cortex (AI) of the cat: frequency tuning and responses to intensity. J Neurophysiol 45:4858.
Phillips DP, Irvine DRF (1983) Some features of binaural input to single neurons in physiologically defined area AI of cat cerebral cortex. J Neurophysiol 49:383395.
Phillips DP, Orman SS, Musicant AD, Wison GF (1985) Neurons in the cats primary auditory cortex distinguished by their responses to tones and wide spectrum noise. Hear Res 18: 7386.[ISI][Medline]
Reale RA, Kettner RE (1986) Topography of binaural organization in primary auditory cortex of the cat: effects of changing interaural intensity. J Neurophysiol 56:663682.
Reser DH, Fishman YI, Arezzo JC, Steinschneider M (2000) Binaural interactions in primary auditory cortex of the awake macaque. Cereb Cortex 10:574584.
Rodrigeus-Dagaeff C, Simm G, de Ribaupierre Y, Villa A, de Ribaupierre F, Rouiller EM (1989) Functional organization of the ventral division of the medial geniculate body of the cat: evidence for a rostro-caudal gradient of response properties and cortical projections. Hear Res 39:103126.[ISI][Medline]
Rouiller EM, Simm GM, Villa AEP, de Ribaupierre Y, de Ribaupierre F (1991) Auditory corticocortical interconnections in the cat: evidence for parallel and hierarchical arrangement of the auditory cortical areas. Exp Brain Res 86:483505.[ISI][Medline]
Sanes DH, Malone BJ, Semple MN (1998) Role of synaptic inhibition in processing of dynamic binaural level stimuli. J Neurosci 18:794803.
Schreiner CE, and Mendelson JR (1990) Functional topography of cat primary auditory cortex: distribution of integrated excitation. J Neurophysiol 64:14421459.
Schreiner CE, Mendelson JR, Sutter ML (1992) Functional topography of cat primary auditory cortex: representation of tone intensity. Exp Brain Res 92: 105122.[ISI][Medline]
Semple MN, Kitzes LM (1993a) Focal selectivity for binaural sound pressure level in cat primary auditory cortex: two-way intensity network tuning. J Neurophysiol 69:462473.
Semple MN, Kitzes LM (1993b) Binaural processing of sound pressure level in cat primary auditory cortex: evidence for a representation based on absolute levels rather than interaural level differences. J Neurophysiol 69:449461.
Smith PH, Populin LC (2001) Fundamental differences between the thalamocortical recipient layers of the cat auditory and visual cortices. J Comp Neurol 436:508519.[ISI][Medline]
Sutter ML, Schreiner CE (1995) Topography of intensity tuning in cat primary auditory cortex: single-neuron versus multiple-neuron recordings. J Neurophysiol 73:190204.
Sutter ML, Schreiner CE, McLean M, OConnor KN, Loftus WC (1999) Organization of inhibitory frequency receptive fields in cat primary auditory cortex. J Neurophysiol 82:23582371.
Volkov IO, Galaziuk AV (1991) Formation of spike response to sound tones in cat auditory cortex neurons: interaction of excitatory and inhibitory effects. Neuroscience 43:307321.[ISI][Medline]
Volkov IO, Galaziuk AV (1992) Peculiarities of inhibition in cat auditory cortex neurons evoked by tonal stimuli of various durations. Brain Res 91:115120.
Wallace MN, Kitzes LM, Jones EG (1991) Intrinsic inter- and intralaminar connections and their relationship to the tonotopic map in cat primary auditory cortex. Exp Brain Res 86:527544.[ISI][Medline]
Weliky M, Kandler K, Fitzpatrick D, Katz LC (1995) Patterns of excitation and inhibition evoked by horizontal connections in visual cortex share a common relationship to orientation columns. Neuron 15:541552.[ISI][Medline]
Winer JA, Diehl JJ, Larue DT (2001) Projections of auditory cortex to the medial geniculate body of the cat. J Comp Neurol 430:2755.[ISI][Medline]
Winguth SD, Winer JA (1986) Corticocortical connections of cat primary auditory cortex (AI): laminar organization and identification of supragranular neurons projecting to area AII. J Comp Neurol 248:3656.[ISI][Medline]
Wu SH, Oertel D (1986) Inhibitory circuitry in the ventral cochlear nucleus is probably mediated by glycine. J Neurosci 6:26912706.[Abstract]