The Influence of Single VB Thalamocortical Impulses on Barrel Columns of Rabbit Somatosensory Cortex

Harvey A. Swadlow1 and Alexander G. Gusev1,2

 1Department of Psychology, The University of Connecticut, Storrs, Connecticut 06269; and  2Moscow Brain Research Institute, Russian Academy of Medical Sciences, Moscow 103064, Russia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Swadlow, Harvey A. and Alexander G. Gusev. The Influence of Single VB Thalamocortical Impulses on Barrel Columns of Rabbit Somatosensory Cortex. J. Neurophysiol. 83: 2802-2813, 2000. Extracellular recordings were obtained from single neurons in ventrobasal (VB) thalamus of awake rabbits while field potentials were recorded at various depths within topographically aligned and nonaligned barrel columns of somatosensory cortex (S1). Spike-triggered averages of cortical field potentials were obtained following action potentials in thalamic neurons. Action potentials in a VB neuron elicited a cortical response within layer 4 with three distinct components. 1) A biphasic, initially positive response (latency <1 ms) was interpreted to reflect activation of the VB axon terminals (the AxTP). This response was not affected by infusion of an alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist within the barrel. In contrast, later components of the response were completely eliminated and were interpreted to reflect focal synaptic potentials. 2) A negative potential [focal synaptic negativity (FSN)] occurred at a mean latency of 1.65 ms and lasted ~4 ms. This response had a rapid rise time (~0.7 ms) and was interpreted to reflect monosynaptic excitation. 3) The third component was a positive potential (the FSP), with a slow rise time and a half-amplitude duration of ~30 ms. The FSP showed a weak reversal in superficial cortical layers and was interpreted to reflect di/polysynaptic inhibition. The amplitudes of the AxTP, the FSN, and the FSP reached a peak near layer 4 and were highly attenuated in both superficial and deep cortical layers. All components were attenuated or absent when the cortical electrode was missaligned from the thalamic electrode by a single cortical barrel. Deconvolution procedures revealed that the autocorrelogram of the presynaptic VB neuron had very little influence on either the amplitude or duration of the AxTP or the FSN, and only a minor influence (mean, 11%) on the amplitude of the FSP. We conclude that individual VB thalamic impulses entering a cortical barrel engage both monosynaptic excitatory and di/polysynaptic inhibitory mechanisms. Putative inhibitory interneurons of an S1 barrel receive a highly divergent/convergent monosynaptic input from the topographically aligned VB barreloid, and this results in sharp synchrony among these interneurons. We suggest that single-fiber access to disynaptic inhibition is facilitated by this sharp synchrony, and that the FSP reflects a consequent synchronous wave of feed-forward inhibition within the S1 barrel.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Barrel columns of somatosensory cortex receive a dense, topographically precise input from a few hundred neurons of the corresponding ventrobasal thalamic barreloid (Jensen and Killackey 1987; Land et al. 1995; Van der Loos 1976). This thalamic input exerts a powerful influence on many cortical neurons, mediating salient receptive field properties and short-latency responses to peripheral stimulation (Armstrong-James et al. 1992; Simons and Carvell 1989; Swadlow 1995). Extracellular cross-correlational methods have been the major tools used to study unitary thalamocortical interactions, and these methods have allowed identification of the classes of cortical neurons that respond monosynaptically to thalamocortical input (Reid and Alonso 1995; Swadlow 1995; Tanaka 1983). Such methods are well-suited for the detection of monosynaptic, excitatory, suprathreshold effects on individual postsynaptic neurons. However, they are less well suited for the detection of inhibitory or disynaptic events (cf., Aertsen and Gerstein 1985), and they do not readily yield information concerning the spatial distribution of events mediated by single thalamocortical afferents.

Spike-triggered averaging of postsynaptic potentials has been used to examine the time course and amplitude of unitary postsynaptic potentials elicited by single presynaptic fibers (e.g., Kirkwood and Sears 1982a; Mendell and Henneman 1971; Munson and Sypert 1979). Spike-triggered averaging can also be used to examine extracellular fields elicited by single presynaptic elements. This method has been predominantly used with large-diameter axons entering the spinal cord, to trace the influence of sensory afferent fibers (Munson and Sypert 1979), descending central axons (Kirkwood 1995; Taylor et al. 1977), or spinal interneurons (Kirkwood et al. 1993; Schmid et al. 1993). In the present work we apply this method to awake sensory neocortex, to trace spatial and temporal synaptic influences of individual ventrobasal thalamocortical axons on topographically aligned barrel columns. We present evidence that, in addition to providing a powerful monosynaptic excitatory input, individual impulses of single thalamic afferents gain access to potent inhibitory mechanisms of sensory neocortex.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Recordings were obtained from S1 cortical barrel columns and from ventrobasal (VB) thalamic barreloids of adult, fully awake Dutch-belted rabbits. All recordings were obtained from the representation of medium-to-large central or posterior vibrissae. Some of the methods have been recently described (Swadlow 1995; Swadlow et al. 1998) and are reported briefly here.

General surgical methods

Initial surgery was performed under pentobarbital sodium anesthesia (initial dose, 25-35 mg/kg) using aseptic procedures. After removal of skin and fascia above the skull, the bones of the dorsal surface of the skull were fused together using stainless steel screws and acrylic cement. A stainless steel rod (6 mm diam, thinned to 2 mm in places to conserve space on the skull) was oriented in a rostrocaudal direction, positioned over the midsagittal suture, and cemented to the acrylic mass. The rabbit was rigidly held by this bar during later surgery and recording sessions. A layer of silicone rubber always covered the exposed skull between recording sessions and was also used to buffer the wound margins from the acrylic cement on the skull. After each recording session the entrance in the skull was filled with gelfoam that was soaked in an antibiotic ointment and capped with acrylic cement. The remaining exposed skull was then treated with antibiotic ointment and covered with a thick layer of the silicone rubber.

Extracellular recording procedures

Methods ensuring the humane treatment of our subjects during immobilization of the head and single-unit recording have been described (Swadlow 1989, 1995). After the above initial surgery, all subsequent recordings were obtained from fully awake rabbits. Rabbits were held snugly within a body stocking from which the head protruded and were placed on a foam rubber pad. The steel bar on the head was then fastened to a restraining devise in a manner that minimized stress on the neck when the head was immobilized. After each recording session the small entrance in the skull (see Achieving topographical alignment between VB barreloids and S1 barrels) was resealed with moist gelfoam that was impregnated with antibiotic ointment and covered with acrylic cement. It is noteworthy that, although rabbits may respond to gentle tactile stimulation of the facial region with movements of the nose or vibrissae, no such responses are observed during the above procedures. Rabbits did not appear disturbed by these manipulations.

Recording from VB thalamocortical neurons

Well-isolated extracellular action potentials were recorded from VB thalamic neurons using quartz-filament microelectrodes of 40 µm OD (Reitboeck 1983) (Thomas Recording). Filaments had an inner core (12 µm diam) of Platinum (90%)/Tungsten (10%) and were pulled to a fine taper in the helium-filled chamber of a vertical puller. Electrodes were either ground to a sharp tip using a diamond grinding wheel, or metal tips were exposed by crushing the very tip of the tapered quartz glass under manual control. Electrode impedance (at 1,000 Hz) was generally 1-2 MOmega .

Five or seven of these quartz-filament electrodes were positioned in a concentric array above the vibrissa representation of VB thalamus. Each electrode was guided within a stainless steel cannula (160-200 µm OD, 100-125 µm ID). The guide tubes were bound together in a concentric organization (total overall OD at the tips, 480-600 µm), and a miniature screw-driven microdrive was attached to each electrode, allowing full independence of vertical motion. The seven cannulae were chronically implanted so that the tips were 2-3 mm above the VB representation of the medium-large central or posterior vibrissae after initial physiological mapping of this structure (Swadlow 1995). On the day of implantation, each cannula contained a stylus (a tungsten rod, 80-100 µm diam), and the tips were sealed with a small amount of melted bone wax. Once the cannula were cemented into place above VB thalamus, styluses were removed and microelectrodes were introduced into each cannula after being lightly coated with antibiotic ointment. Microdrives were then attached to the microelectrodes, and they were driven into the very superficial portion of VB thalamus. This nucleus was physiologically identified by 1) the presence of reliable, short-latency (<6 ms) responses to stimulation of a single, dominant vibrissa (the "principal vibrissa") (Simons 1978), and weaker or no responses to stimulation of surrounding vibrissae, and 2) at least some neurons that showed a strong preference for direction of whisker displacement. After this identification procedure, microelectrodes were then retracted ~200 µm and remained in this position for several days until cortical mapping was achieved.

After preamplification and filtering (low- and high-frequency 1/2 band-pass, 300 and 10,000 Hz, respectively), VB spike data were led to a digital oscilloscope for display and analysis of synaptic or antidromic latencies. Spike data were also digitized at 25-33 kHz, and action potentials were discriminated on-line, using a commercially available software package (DataWave Technologies). This package allows spikes to be sorted based on a number of parameters derived from voltage amplitude and time measures. Spike waveforms were saved, along with stimulus information, and were subsequently reexamined and discriminated off-line. Only well-discriminated neurons were selected for analysis in this study.

In some cases stimulating electrodes were inserted into VB through two of the cannulae. This allowed antidromic identification of layer-6 corticothalamic neurons recorded via the cortical recording electrode.

Cortical recordings of field potentials

Cortical recordings were obtained using several different procedures.

1) Quartz-Platinum fiber electrodes were similar to those used in thalamus. However, these electrodes were fabricated from larger diameter fibers so that the dura could be readily penetrated (80 µm OD, 25 µm metal core) and were tapered and sharpened so that impedances were usually 0.6-1 MOmega . In some cases, one to seven of these electrodes were acutely placed within barrel cortex using a motor-driven, seven-channel microdrive (tip separations, 200-300 µm) (Eckhorn and Thomas 1993). In other cases, they were chronically implanted within the cortex, being guided by a three- to seven-barrel cannula system cemented to the skull and positioned just above the dura. Independent vertical movements within the cortex were achieved using the same screw-driven microdrive system described above for thalamic recordings.

2) Silicone probes1 with six independent recording surfaces (vertically separated by 200 µm) were placed within the cortex and were lowered until the deepest recording site reached the approximate depth of layer 6. Stimulation pulses were then delivered via VB stimulating electrodes (above) until a long-latency (>10 ms) antidromically activated neuron was detected that showed a supernormal period of increased conduction velocity (decreased antidromic latency) at intervals of 8-12 ms after a prior impulse (Swadlow 1989). Antidromic identification was based on 1) a refractory period of <2 ms and 2) a constant latency (<0.2 ms variability) to the second of two pulses delivered at an interval of 10 ms (Swadlow et al. 1978). Previous work has shown that such neurons are found exclusively in layer 6, and that descending corticofugal neurons of layer 5 have more rapidly conducting axons and do not show supernormal conduction (Swadlow 1989, 1990).

Field activity was continuously recorded from cortical sites at sampling frequencies of 7,000-8,000 Hz. After amplification and filtering (half-amplitude low- and high-frequency responses, 10 and 9,000 Hz, respectively), field data were digitized and led to DataWave data acquisition system. Spike-triggered averages of field data with VB triggering spikes were created off-line using our own software. These were generated by averaging the cortical responses to >= 3,000 VB spikes. No stimuli were delivered, and animals sat in a quiescent state. We rejected field data in which high-frequency, high-voltage transients occurred.

Achieving topographical alignment between VB barreloids and S1 barrels

Our general experimental strategy was to 1) record from one or more superficial barreloids of VB thalamus and identify the principal vibrissa providing dominant input, 2) identify the topographically aligned cortical barrels, and 3) localize layer 4 using physiological criteria and record spike-triggered averages. 4) Next, the VB electrodes would be lowered further into a new set of more ventral VB barreloids, topographic alignment with the appropriate new set of cortical barrels would be reestablished, and new averages would be generated. Several steps were necessary to achieve these aims.

IDENTIFYING THE PRINCIPAL VIBRISSA PROVIDING INPUT TO VB BARRELOID NEURONS UNDER STUDY. Initial assessment of the principal vibrissa for each VB neuron was done qualitatively, using hand-held stimuli. In most cases, deflection of only a single vibrissa resulted in a strong response, and other whiskers yielded considerably weaker or no responses. For those neurons showing strong responses to deflection of >1 whisker, latency measures were obtained (below), and the whisker yielding the shorter latency was considered the principal vibrissa.

IDENTIFYING THE TOPOGRAPHICALLY ALIGNED S1 BARREL. To minimize the amount of dura exposed during the search for topographical alignment (because exposed dura gradually thickens and microelectrode penetrations become difficult), it was necessary to make microelectrode penetrations through very small openings in the skull. This was achieved by using a dental drill to thin a region of skull (~3 × 3 mm) above the "barrel field" of S1 to a thickness of ~200 µm (from an initial thickness of 1.5-2 mm). At this thickness the skull was transparent, and the larger superficial vessels could be clearly visualized. Thereafter, very small entrances in the skull could be made using a dental drill with a tip diameter of 0.24 mm. In the initial entrance, a finely tapered, low-impedance (<1 MOmega ) tungsten-in-glass microelectrode was then inserted to the approximate position of layer 4. This layer was physiologically identified by the presence of a strong, short-latency (<7.0 ms), multiple-unit response to very brief (<4 ms) air-puff stimulation of the vibrissae (Swadlow 1995; Swadlow et al. 1998). Next, the principal vibrissa for this electrode site was ascertained as described above for VB neurons. Because of the orderly representation of the vibrissae on the cortical surface, only a few such penetrations were usually necessary to locate the S1 barrel that was topographically aligned with the VB neuron under study. In some cases, strong, short-latency (<7 ms) responses were observed from each of two vibrissae, probably reflecting a transition region between two barrels. In these cases, the electrode was moved to a position resulting in a dominant, short-latency input from single vibrissa (presumably nearer the center of the barrel).

FIELD RECORDING FROM IN OR NEAR LAYER 4. After topographical alignment, the tungsten microelectrode used for initial mapping was replaced with a quartz-platinum electrode (or silicone probe electrode) used to record spike-triggered averages. The identity of the principal vibrissa was re-ascertained and the depth of this electrode was adjusted to maximize short-latency (<7 ms) multiple-unit responses to deflection of the principal vibrissa (above). In some cases additional recording electrodes were placed in neighboring barrels.

NEW VB NEURONS WERE ISOLATED. During subsequent recording sessions thalamic electrodes were lowered until new VB neurons were isolated. Principal vibrissae were ascertained and topographic alignment was reestablished with cortical recording sites.

Antagonizing alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors near the recording site

To block postsynaptic components of the unitary field potential 6,7-dinitroquinoxaline-2,3-dione (DNQX) was pressure injected into the cortex at a distance of 350 µm from cortical recording sites. DNQX (1 µl, 5 mM) was dissolved in 165 mM NaCl and injected within or near layer 4 over a period of 6 min, using a 36-gauge (112 µm OD, 60 µm ID) sharpened cannula.2


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In three rabbits, 25 VB neurons were studied that were in precise topographic alignment with a cortical field electrode. Twenty of these VB neurons elicited an average unitary field potential with a clear short-latency (<1 ms) response within the topographically aligned barrel column. These 20 neurons provide the basis for most of the present analyses. Another data set obtained from two additional rabbits consisted of 13 VB neurons that each elicited a short-latency response within the topographically aligned S1 barrel column. These cells were used to study the cortical depth profiles of the elicited field potentials.

Figure 1 presents the spike-triggered average obtained from an S1 recording site following spontaneous impulse activity in a topographically aligned VB neuron. For this recording site, and for each of the topographically aligned sites studied here, the response profile within and near layer 4 could be readily divided into three distinct components: 1) an initially positive, biphasic or triphasic response thought to represent the invasion of the presynaptic impulse into the axon terminal arborization within layer-4 (the axon terminal potential, AxTP), 2) a longer-latency, negative-going potential believed to reflect a local, focal synaptic depolarization (the focal synaptic negative potential, FSN), and 3) a subsequent long-lasting positive potential, believed to reflect focal synaptic hyperpolarization (the focal synaptic positive potential, FSP). Table 1 presents the latency, amplitude, and temporal characteristics of these responses. Values given in the table (and, with noted exceptions, those presented below) are for only those 17 topographically aligned thalamocortical sites that yielded both AxTPs and FSNs of >1 µV.



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Fig. 1. A: spike-triggered average waveform elicited in a cortical barrel resulting from spontaneous impulse activity in a topographically aligned ventrobasal (VB) barreloid neuron. Left and right arrows indicate the onset of the axon terminal potential (AxTP) and focal synaptic negativity (FSN), respectively. B: same spike-triggered average, taken at a slower sweep speed, showing the time course of the focal synaptic positivity (FSP). C: for 8 VB neurons, the relationship between antidromic latency to cortical stimulation and latency of the AxTP component of the unitary field potential.


                              
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Table 1. Values obtained from 17 aligned cases in which the amplitude of both the AxTP and the FSN exceeded 1 µA

AXTP

The AxTPs had latencies of 0.54-0.92 ms (mean, 0.71 ms). These latencies were measured at the onset of the initial positive deflection, at a depth producing the maximal amplitude cortical response (left arrow in Fig. 1A). Maximum amplitudes of the AxTP were always found at cortical depths in which minimal latencies (<7.0 ms) were observed in multiple-unit cortical responses after stimulation of the receptor periphery. These maximal amplitude responses were therefore believed to be in or very near to layer 4 (METHODS) (Swadlow 1995; Swadlow et al. 1998). The maximal amplitudes of the AxTP (measured from the peak of the initial positive response to the peak of the secondary negative response) had a mean value of 3.25 µV, and the duration of the initial positive peak (taken at half amplitude) was 0.23 ms.

Eight of the VB neurons generating spike-triggered averages were tested with microstimulation pulses (0.2 ms, <20 µA cathodal) delivered to the cortical recording site. In each case this resulted in an antidromic response. Figure 1C shows that the antidromic latency was correlated with the latency of the AxTP (R2 = 0.82, P = 0.002).

In three cases, 1 µl of DNQX were injected into the cortex at distances of 300-400 µm from the recording site.

Figure 2 shows the results of injecting 1 µl of DNQX within the cortex at a distance of 350 µm from the recording site. AxTPs were little affected by antagonism of AMPA/kainate receptors within the cortical barrel (Fig. 2A). In contrast, the FSN and FSP were nearly abolished by this manipulation (Fig. 2B) and took several hours to reach partial recovery (Fig. 2C). Virtually identical experiments were performed in two additional cases (separation between recording microelectrode and injection site, 300-400 µm), with nearly identical results. In each of these two additional cases, the amplitude of the AxTP following the injection remained very close (85-105%) to the control amplitude However, after the injection of DNQX, the amplitudes of the FSNs and FSPs were reduced to <20% of control values.



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Fig. 2. Results of injecting 1 µl of 6,7-dinitroquinoxaline-2,3-dione [DNQX; an alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist] within the cortex at a distance of 350 µm from the recording site. A: control, before injection. B: 18 min after injection of DNQX 350 µm from the recording site. Only the AxTP remains. C: partial recovery, 150 min after the injection.

FSN

The FSN was sharply rising (10-90% rise time, 0.68 ms) and emerged out of the trailing edge of the AxTP (arrow at right in Fig. 1A). The latency of this response was, in some cases, difficult to ascertain precisely because the FSN arose out of the AxTP, and, as noted above, this latter response varied in both amplitude and waveform with cortical depth. Nevertheless, we estimated the onset of the FSN to have a mean latency of 1.67 ms and, for individual pairs, to be roughly correlated with the latency of the AxTP (R2 = 0.24, P = 0.04).3 The maximal amplitude of the FSN (measured from the baseline to the peak of the negative potential) had a mean value of 3.86 µV. Generally, the FSN decreased to the baseline level within several milliseconds. The duration of the half-amplitude response had a mean value of 2.25 ms.

FSP

The FSN was followed by a prolonged positive response, the FSP (Fig. 1B). The onset of this response appeared to emerge from the trailing end of the FSN. Because of this, and because of the relatively long time course of this response, we make no attempt to measure the latency of onset. The maximal amplitude of the FSP (measured from the baseline just before the thalamic spike to the peak of the positive potential) had a mean value of 5.42 µV. The FSP had a total duration of 30-70 ms and a mean half-amplitude duration of 31.8 ms.

Depth distribution of field components

The maximal amplitudes of the AxTP, FSN, and FSP were all found within or very near to layer 4. Figure 3 presents a case in which a single stationary probe with six recording sites was used to generate spike-triggered averages at vertical site separations of 200 µm. The maximal response (Max) for all unitary field components was seen at site 3 (from top), and at sites 3 and 4, multiple-unit responses to peripheral stimulation were seen at latencies of <7 ms, indicating that these sites were within or very near to layer 4. The deepest site (bottom) was located within layer 6. This assertion is based on the presence of a neuron recorded at this site that showed 1) a very long latency (>10 ms) antidromic response to thalamic stimulation and 2) a decrease in antidromic latency (the supernormal period) after a single prior impulse (METHODS). Although the latency of the AxTP seen in Fig. 2 is approximately the same at each of the six sites, the amplitude reaches a clear peak at site 3 and is diminished both above and below this depth. The amplitude of the FSN and FSP also reaches a peak at the same site. Figure 4 presents the relationship between cortical depth and amplitude of the AxTP, the FSN, and the FSP for six VB neurons studied with the same six-channel stationary silicone probe described above. Note that for each of these VB neurons, the amplitude of the AxTP, the FSN, and the FSP reached a peak at or next to site 3 (Max) and was considerably diminished both superficially and deeper within the cortex.



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Fig. 3. A: single probe with 6 recording sites (vertical distance between sites, 200 µm) was chronically implanted into an S1 barrel column. Spike-triggered averages were elicited by a neuron in the topographically aligned VB barreloid. Maximal amplitude potentials were seen in the 3rd trace from the top (Max). B: same traces, but at a slower sweep-speed (horizontal cal bar, 4 ms in A, and 40 ms in B and C. C: spike-triggered averages simultaneously generated at the same recording sites from a VB barreloid neuron that was missaligned by 2 barreloids. Vertical lines indicate the time of the thalamic triggering spike.



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Fig. 4. Relationship between cortical depth (x-axis) and amplitude (y-axis) of the AxTP (left), the FSN (middle), and FSP (right) for 6 VB neurons studied with the same 6-channel stationary silicone probe described above. Note that the amplitudes of all 3 components of the unitary field potential reached a peak at intermediate cortical depths, and diminished considerably both superficially and deeper within the cortex.

No signs of reversal of any components of the unitary field potentials were revealed by the data presented above. However, the above recordings extended only ~ 400 µm superficial to the site of the maximal response, and this may have been insufficient to reveal the reversal. Therefore 13 additional VB neurons were studied, each with a well-defined AxTP, FSN, and FSP within layer 4 of the topographically aligned barrel column. We used a traditional, finely tapered microelectrode with a single low-impedance (<1 MOmega ) recording site to sequentially record spike-triggered averages from different depths (200-300 µm vertical separation of recording sites) within the barrel column. In no case did the AxTP or the FSN show any sign of reversal in more superficial cortex (up to 900 µm superficial to the maximal response). However, in each of the 13 cases the FSP showed a weak reversal at distances of 400-900 µm superficial to the maximal response. Figure 5 shows responses obtained from one such case. Here, the maximal response of all unitary field components occurred at a depth of 1,115 µm beneath the surface of the dura (bottom), and a mild reversal of the FSP was seen at a depth of 415 µm. Note that for this case (and for each of the 13 cases) the maximum amplitude of the reversed FSP was much less (17 ± 7%, mean ± SD) than the maximum value of the FSP seen near layer 4. 



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Fig. 5. Unitary field potentials elicited by a VB neuron at various depths within the topographically aligned barrel column. Numbers to left indicate depth (µm) beneath the surface of the dura at which the record was obtained. In this case, the maximal response for all components of the potential was seen at a depth of 1,115 µm beneath the surface of the dura (bottom). At a depth of 415 µm a weak reversal of the FSP is seen, but not of the AxTP or FSN.

Responses elicited by VB neurons of nonaligned barreloids

Responses from nonaligned barreloid-barrel pairs were generally studied by identifying a cortical site that yielded a clear AxTP and FSN to spikes of a topographically aligned VB barreloid neuron and, in addition, recording fields from this site that were simultaneously generated by neurons in one or more neighboring VB barreloids. In this way we could be confident that a weak unitary field potential generated by the nonaligned VB neuron was not due to a nonoptimal depth placement of the cortical recording site. Figure 3C shows one such case. Here, the same six-channel silicone probe electrode that generated the average unitary waveforms shown in Fig. 3, A and B (from an aligned VB barreloid neuron), also, during the same recording session, generated the average unitary waveforms from a VB barreloid neuron that was missaligned by two barreloids. Note that virtually no AxTP or FSN is seen, and only a small hint of an FSP is detectable. Figure 6 shows a histogram of the amplitude of the AxTP, FSN, and FSP when triggering VB neurons are in topographically aligned barreloids,4 or when they are missaligned by one, or by or two to three barreloids. Note that all components of the spike-triggered average are strongly attenuated when missaligned by a single barreloid.



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Fig. 6. Histogram of the amplitude of the AxTP, FSN, and FSP when triggering VB neurons are in topographically aligned barreloids, or when they are missaligned by 1, or by 2 or 3 barreloids. Note that the AxTPs and FSNs are strongly attenuated when missaligned by 1 barreloid, and virtually absent when missaligned by 2 or 3 barreloids. Error bars indicate SD.

In one fortuitous case, three thalamic neurons (a1, a2, and b1) in two neighboring VB barreloids (a and b) were recorded simultaneously with fields in two neighboring cortical barrels (A and B), each of which was in topographic alignment with one of the VB barreloids under study, and missaligned with the other. Figure 7 (top) presents this case schematically, and spike-triggered averages obtained from sites A and B are shown below. Note that VB neurons a1 and a2 elicited clear AxTPs and FSNs in S1 barrel A (aligned), but not in barrel B (neighboring barrel, nonaligned). Conversely, the VB neuron "b" elicited clear responses in S1 barrel B, but not in barrel A. 



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Fig. 7. Top: schematic illustration of a case in which 2 thalamic electrodes recorded from neurons in neighboring VB barreloids (a and b), and cortical electrodes recorded spike-triggered averages in the topographically aligned (and neighboring) S1 barrels (A and B). Spike-triggered averages were generated by 2 neurons in barreloid a (a1 and a2) and by 1 neuron in barreloid b (b). Spike-triggered averages elicited in barrel A by barreloid neurons a1, a2, and b are shown at bottom left. Spike-triggered averages elicited in barrel B by the same 3 VB neurons is shown at bottom right.

Contribution of the autocorrelogram to spike-triggered averages

In all methods of spike-triggered averaging, the autocorrelogram of the triggering spike can influence the average waveform obtained (Kirkwood 1979; Moore et al. 1970) (see DISCUSSION). We assessed the contribution of the autocorrelogram by deconvolution of each of the spike-triggered averages that contributed to Table 1 with the associated autocorrelogram of the triggering VB spike (Brillinger et al. 1976; also see Eggermont et al. 1993).5 For each of the 17 cases, the deconvolved AxTP and FSN were very similar to the original waveforms (within ±10% in amplitude and duration). The effect of the deconvolution on the FSP was somewhat greater and depended on the shape of the autocorrelogram. Whereas sharp side bands in the autocorrelogram resulted in a decrease in the amplitude of the deconvolved FSP, a central trough resulted in an increase in the amplitude of the FSP. For most of the 17 cases, the change in the amplitude of the FSP was <15% (mean change, 13 ± 7.4%), but in a few cases, greater changes were seen (up to 28%). Figure 8 shows 3 of the 17 cases. Extreme cases are shown on the left (very sharp side-bands in the autocorrelogram) and the right (deep trough in the autocorrelogram), where the magnitude of the FSP varied by >20%. To validate the Fourier-based deconvolution procedure, we convolved the results of the three deconvolutions shown in Fig. 8 with their associated autocorrelograms. In each case, the original waveform was reconstituted and was virtually indistinguishable from the original spike-triggered average waveform.



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Fig. 8. A: autocorrelograms of 3 VB neurons that yielded well-defined spike triggered averages. Whereas the central autocorrelogram is relatively flat, the one on the left shows very strong side bands (indicating many short interspike intervals), and the one on the right shows a central trough. Peaks at the very center of the autocorrelograms have been eliminated. B: associated spike-triggered average waveform (- - - - -) presented at a fast time base to show the AxTP and FSN. ---, results after deconvolution of the spike-triggered average waveform with the associated autocorrelogram (above). C: same waveforms as in B, but shown at a slower time base to show the FSP.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present work has identified three components in the average unitary field potential elicited in an S1 barrel by a single, topographically aligned VB barreloid neuron.

AxTP

Previous studies of extracellular spike-triggered averages in spinal cord and brain stem have differentiated the presynaptic component of the potential into "axonal" and "terminal" components (Kirkwood 1995; Munson and Sypert 1979; Schmid et al. 1993). Although we observed a low-amplitude axonal potential throughout the depth of the cortex, we have focused here on the larger amplitude component found in the vicinity of layer 4. The maximal amplitude response at this depth, as well as the restriction of the AxTP to a single barrel column were expected, based on morphological studies of individual VB axon terminal arborizations (Jensen and Killackey 1987).

The initially positive-going response of the AxTP is consistent both with previous analyses of axon terminal responses and with theoretical analyses of recordings from near the sealed ends of axons (cf., discussion in Munson and Sypert 1979). The total duration of the initial positive component was brief (mean, 0.37 ms). It should be noted, however, that the timing of the triggering VB spike was accurate only to the nearest 100 µs (because of software limitations), and this will result in an overestimation of the duration of any resultant waveforms by a similar value. By testing our system using calibrated square waves, this was found to be the case. It should also be noted that the duration of the AxTP was measured at a depth that yielded a maximal amplitude waveform, and this was thought to correspond to layer 4. In some cases, the duration of the AxTP at this depth was somewhat greater than observed in axonal potentials of reduced amplitude seen 200-500 µm deeper in cortex (e.g., Fig. 3A). The increased amplitude and duration of the AxTP within layer 4 is consistent with increased membrane area and temporal delays occurring in a highly branched terminal axon.

The AxTP occurred at a mean onset latency of 0.71 ms after the onset of the thalamic action potential. This value is in agreement with a mean antidromic latency 0.91 ms reported for VB neurons that were antidromically activated via S1 stimulation (Swadlow 1995), indicating a mean axonal conduction time of ~ 0.71 ms [axonal conduction time was estimated by subtracting 0.2 ms from the antidromic latency (e.g., Fuller and Schlag 1976)].

Antidromic responses were seen in each of eight tested VB neurons that were in topographic alignment with a cortical recording site, and antidromic latency was highly correlated with the latency of the AxTP (Fig. 1C). For these cells, however, antidromic latencies were a mean of 0.38 ms greater than were AxTP latencies. Approximately 0.2 ms of this discrepancy is expected, due to utilization time after the antidromic stimulus and delay of invasion near the soma (e.g., Fuller and Schlag 1978), and 50 µs can be attributed to the time stamp resolution of the VB triggering spike (100 µs, above). The reason for the remaining discrepancy (0.13 ms) is unknown.

FSN

Several lines of evidence favor the hypothesis that the FSN is largely due to local active inward currents associated with monosynaptic excitation via the VB thalamocortical neuron under study: 1) the amplitude of the negative potential was sharply delimited in depth; 2) this optimal depth was the same as that which yielded a) maximal amplitude AxTPs, b) very-short latency (<7 ms), multiunit spike activity elicited by peripheral air-puff stimulation, and c) the maximal amplitude short-latency evoked response to peripheral air-puff stimulation; 3) the FSN was sharply limited in the horizontal domain, with little response being seen in the neighboring barrel; 4) the latency of onset of the FSN is consistent with monosynaptic thalamocortical activation and was significantly correlated with the latency of the AxTP; and 5) the FSN was completely blocked by antagonism of AMPA/kainate receptors, which left the AxTP intact. Together, these data strongly suggest that the FSN reflects local inward currents associated with monosynaptic thalamocortical excitation, and that the focus of this activity is within layer 4.

The FSN had a sharp rise time and a total duration of ~4 ms. Although Kirkwood (1995) reports similarly brief extracellular focal synaptic potentials in bulbospinal neurons of brain stem, Munson and Sypert (1979) found that extracellular potentials elicited by 1a afferent fibers mirror the time course of motoneuron EPSPs. As noted above, it was not possible to differentiate the end of the FSN from the beginning of the FSP. It is likely that the falling phase of the FSN (which was often abrupt, e.g., Fig. 1) reflects the onset of disynaptic inhibition (Agmon and Connors 1992; Swadlow 1995), and this may have truncated the FSN.

Cross-correlation studies of VB thalamocortical neurons and putative feed-forward inhibitory interneurons of S1 (Swadlow 1995) revealed that a sharp increase in cortical spike probability occurs from 1.4 to 2.0 ms following VB action potentials (Swadlow 1995). This peak in the cross-correlogram is very brief, having a half-amplitude response of ~1 ms. These values closely coincide with the rising phase and peak of the FSN reported in the present work. The temporal correspondence between the rising phase of the FSN and the time course of spikes elicited by VB thalamocortical neurons supports our contention that the FSN is due to excitatory synaptic currents and also supports the hypothesis that spikes resulting from monosynaptic input are largely generated during the rise time or derivative of the excitatory postsynaptic potential (EPSP) (Cope et al. 1987; Kirkwood and Sears 1982b). Although synaptic currents are generally thought to be the main contributors to cortical field potentials (e.g., Mitzdorf 1985), correspondence between the rising phase of the FSN and the sharp increase in cortical spike probability raises the possibility that local action potentials may make some contribution to the FSN.

FSP

Our data suggest that the FSP reflects an outward current generated by local inhibitory postsynaptic potentials (IPSPs). Two lines of evidence support this notion and argue against the possibility that the FSP is a passive reflection of a distant inward current. 1) The cortical depth yielding the maximal-amplitude FSP was the same as that yielding the maximal amplitude of the AxTP and FSN. As noted above, the short-latency synaptic responses to air-puff stimulation seen at this depth are indicative of layer 4. This layer is rich with GABAergic interneurons (Harris and Woolsey 1983; Simons and Woolsey 1984; White 1978, 1989) that have an axonal projection that is largely restricted to the same layer 4 barrel (Harris and Woolsey 1983). One would therefore expect IPSPs generated by these neurons to have a similar depth distribution to the EPSPs generated by thalamocortical afferents. 2) The amplitude of the FSP reached a sharp peak within and near layer 4, and only a weak reversal of the FSP was seen in superficial cortex. This is a predictable consequence of an active outward current in layer 4, and a passive inward current in superficial cortex. This result is expected because inhibitory interneurons form many of their synapses on pyramidal neurons (White 1989), which have apical dendrites that extend into superficial cortex. It is important to note that a positive-going waveform in layer 4 could result from excitatory input to superficial cortical layers. However, in such a case a stronger negativity would be expected to occur in superficial layers, and a weaker and more spatially dispersed positivity would be expected to occur in deeper layers. For these reasons, we believe that the FSP reflects outward currents associated with local IPSPs, and that the focus of this activity is within layer 4. However, this conclusion must remain tentative until confirmed or disconfirmed by intracellular analyses.

The FSP was somewhat more broadly distributed both horizontally and in depth than either the AxTP or the FSN. This could result if some of the inhibitory interneurons that receive monosynaptic VB input have an axon terminal distribution that is more widespread than that of the thalamocortical axons. Alternatively, because the FSP can be influenced by broadly synchronous activity of presynaptic elements (below), this could reflect a weak, broad synchrony between VB thalamocortical neurons of the same and neighboring barreloids.

Assessing the influence of the autocorrelogram on the obtained spike-triggered averages

A confounding problem for all methods using spike-triggered averaging is that the obtained waveform (the intracellular or extracellular average unitary waveform or the cross-correlogram function) may receive contributions in addition to those initiated by the "triggering" event under direct observation. In this respect, the arguments of Moore et al. (1970) concerning "primary effects" versus "secondary effects" on cross-correlogram functions apply equally well to methods of spike-triggered averages of intracellular and extracellular potentials (also see, e.g., Kirkwood 1979). Thus any cross-correlogram function or spike-triggered average waveform may be considered to be a compound waveform that results from the convolution of the idealized, unitary waveform (the "kernel" waveform) (cf. Kirkwood 1979; Knox 1974)6 with 1) the autocorrelogram of the triggering neuron under study, 2) the cross-correlogram of the triggering neuron with other neurons that project to the neuron or tissue under study (Hamm et al. 1985; Moore et al. 1970), and 3) the poststimulus histogram of any variations in conduction time that result from axonal conduction or synaptic delays.

We used the Fourier-based method of deconvolution (Brillinger et al. 1976; Eggermont et al. 1993) to assess the contribution of the autocorrelogram to the spike-triggered average waveform. This analysis indicated that the autocorrelogram contributed little to either the amplitude or time course of the AxTP or the FSN. In addition, in most of the 17 cases analyzed, we saw only modest changes in the amplitude and time course of the FSP. However, when the autocorrelogram showed either strong central side bands (Fig. 8, left) or a deep central trough (Fig. 8, right) the FSP was significantly influenced.

We did not analyze the influence of variations in conduction along VB thalamic axons on spike-triggered averages because of the uniformly rapid conduction time of this axonal system (Swadlow 1995), and we did not analyze the influence of presynaptic synchrony (Hamm et al. 1985; Moore et al. 1970). The most obvious potential source of such synchrony is between the presynaptic VB neuron under study and other VB neurons in the same barreloid. Whereas sharply synchronous activity (cf., Alonso et al. 1996; Swadlow et al. 1998) among VB neurons could influence the faster components of the spike-triggered average (i.e., the AxTP and the FSN), more broadly synchronous activity could influence the slower components (the FSP). Preliminary studies of VB neurons within the same barreloid have revealed little sharply synchronous activity (Swadlow et al. 1998; present work). However, a definitive analysis of the influence of presynaptic synchrony on thalamocortical unitary field potentials must await further analyses of the degree of synchrony among thalamocortical neurons of the same VB barreloid.

Comparisons of unitary thalamocortical field potentials with previous studies of sensory neocortex

Extracellular field potentials elicited by single thalamocortical afferents have not been previously studied in any region of cortex. In principle, however, they should bear a resemblance to the extracellular fields and underlying postsynaptic potentials generated by synchronous barrages of thalamocortical impulses (i.e., those resulting from sensory or gross electrical stimulation). The earliest synaptic responses in primary sensory cortex following electrical stimulation of thalamic afferents are monosynaptic EPSPs seen predominantly within and near layer 4 (e.g., Agmon and Connors 1992; Ferster and Lindstrom 1983). In the extracellular record, this early response consists of a negative potential that reflects active current sinks (Agmon and Connors 1991; Mitzdorf 1985). This potential usually reverses in very superficial layers, reflecting the passive return of currents (current sources) through the apical dendrites of pyramidal neurons. In the present work, the spatial distribution and other characteristics of the FSN are highly consistent with excitatory synaptic currents being generated within and near to layer 4. However, we found no evidence for the expected reversal of this potential in superficial cortex. This could be due to a domination of the excitatory extracellular synaptic currents by nonpyramidal elements with relatively local dendritic fields. Although thalamocortical afferents to layer 4 of barrel cortex are known to synapse onto spiny stellate, nonspiny stellate, and pyramidal neurons (White 1989), only the latter population would contribute to a reversal of this potential in upper layers. Recent work in the thalamocortical slice preparation shows that fast-spike GABAergic interneurons of layer 4 show large unitary EPSPs with rise times to thalamocortical synapses that are much faster than those of pyramidal neurons (Gibson and Connors 1998). The summation of the extracellular currents generated by the synchronous generation of such EPSPs could contribute heavily to the extracellular field potential elicited by single thalamocortical afferents. Thus extracellular currents generated by these elements and other nonpyramidal neurons may dominate the extracellular record.

Alternatively, the lack of reversal in the FSN in superficial cortex could be due to limitations in the methods used here to analyze unitary field potentials. Current source-density analysis (e.g., Mitzdorf 1985) has not yet been used to study currents generated by unitary presynaptic elements. This method may be well suited to localize such minute currents, but it requires a larger number of vertically spaced sampling points than was used here.

Intracellular studies in a wide range of species and sensory systems have shown that an ubiquitous disynaptic IPSP follows the monosynaptic EPSP that is generated by a synchronous thalamocortical barrage (e.g., Creutzfeldt and Ito 1968; Ferster and Lindstrom 1983; Hellweg et al. 1977). One might suspect that the occurrence of prominent di/polysynaptic IPSPs would require synchronous generation of action potentials, such as those resulting from potent sensory or electrical stimulation. Indeed, previous work examining spike-triggered averages of intracellular postsynaptic potentials or extracellular fields have shown little or no evidence of disynaptic inhibition. In the present work, however, the FSP resulting from single "spontaneous" VB thalamocortical impulses was of greater amplitude and duration than any other component of the spike-triggered average. Unlike the FSN, this response did show a clear-cut reversal in superficial cortex. This is expected because pyramidal neurons are thought to provide the synaptic targets of most inhibitory interneurons of sensory cortex (White 1989). One explanation for the large magnitude of the FSP in barrel cortex is given below.

Current source-density analysis of responses generated by gross sensory or electrical stimulation of afferent pathways has revealed a vertical cascade of excitatory events following initial activation of layer 4 (Mitzdorf 1985). Our single-fiber analysis yields no evidence of such a vertical cascade, with all presumed active current sinks and sources reaching a peak within or near to layer 4. This result is, perhaps, not surprising. A vertical cascade of excitation would require the generation of action potentials in pyramidal or spiny stellate neurons, and single thalamocortical impulses may be rarely sufficient for this task. In contrast, the inhibition reflected by the FSP is thought to result from IPSPs generated by the activation of fast-spike GABAergic interneurons within and near layer 4 (see below). These cells have very low thresholds to excitatory input and do discharge at reasonably high "efficacy" following VB thalamocortical impulses (Swadlow 1995). As noted above, the axons of these neurons are largely restricted to the layer-4 barrel in which they are found. The resulting outward synaptic currents would therefore be expected to reach a peak near layer 4.

Conclusions: effects of individual VB thalamocortical neurons on an S1 barrel

Our results indicate that a single action potential in a single VB thalamocortical neuron results in a clear sequence of events that is largely limited to a single S1 barrel: 1) the arrival of the VB impulse in layer 4 (the AxTP), 2) monosynaptic excitation generated by this axonal input (the FSN), and 3) a long-duration positive wave (the FSP) that we interpret to reflect local di- or poly-synaptic inhibition.

Previous work in rabbit barrel cortex (Swadlow 1995) has identified a class of fast-spiking (Amitai and Connors 1995; Kawaguchi and Kubota 1993; McCormick et al. 1985; Simons 1978), putative feed-forward inhibitory interneurons (suspected inhibitory interneurons, SINs) within and near layer 4. Results derived from cross-correlation and microstimulation indicated that most SINs within a layer-4 barrel receive monosynaptic input from a large number of the VB thalamocortical neurons of the topographically aligned VB barreloid. They are thus engaged in a secure, highly divergent/convergent relationship with neurons of the aligned VB barreloid (a "complete network") (cf. Abeles 1991; Griffith 1963).

A predictable consequence of such divergent/convergent networks is a degree of "sharp" synchrony among the recipient elements (Moore et al. 1970; Sears and Stagg 1976), and SINs within an S1 barrel indeed demonstrate sharp synchrony (Swadlow et al. 1998). Thus when recording the spike trains of two SINs randomly selected within the same barrel, ~4% of the spikes of the two SINs are coincident (±1 ms), creating a sharp peak in the cross-correlogram. Given ~350 SINs in an S1 barrel,7 one may conclude that sharply synchronous events between SINs are not limited to the two cells under study, but will include other SINs as well (the number will depend on the degree of dependence or independence of synchronous events). We have previously argued that the sharply synchronous activity among these elements will result in a corresponding synchronous, feed-forward release of GABA onto postsynaptic targets, and that such summated IPSPs may be especially effective when excitatory drive to target cells is weak and asynchronous (Swadlow et al. 1998). We suggest that the FSPs observed here reflect this sharply synchronous, feed-forward inhibitory process, and that a functional consequence of sharp synchrony among feed-forward GABAergic interneurons is to amplify the impact of individual VB impulses on inhibitory mechanisms of the neocortex.


    ACKNOWLEDGMENTS

This work was supported by National Science Foundation Grant IBN-9723967 and National Institute of Mental Health Grant MH-59322 to H. A. Swadlow and Russian Foundation of Basic Research Grant 98-04-48208 to A. Gusev.


    FOOTNOTES

1 These electrodes were generously provided by the University of Michigan Center for Integrated Sensors and Circuits. The "Schmidt 2" model was used in these experiments.

2 We made several unsuccessful attempts to antagonize postsynaptic components of the spike-triggered average (Kirkwood et al. 1991) by iontophoresis of DNQX (e.g., Swadlow and Hicks 1997). This may be due to the large size of the barrels studied (~300-400 µm diam) and the relatively limited spread of agents by this method.

3 In three cases our estimates of FSN latency were supported by subsequently eliminating the FSN using DNQX (e.g., Fig. 2, A and B).

4 Mean amplitude values of topographically aligned components of the STA are somewhat less in Fig. 6 than in Table 1. This is because Table 1 is derived from only those 17 spike-triggered averages in which the AxTP and FSN exceeded an amplitude of 1 µV. Values here are from all 25 spike-triggered averages obtained from topographically aligned VB-S1 pairs.

5 This is achieved by dividing the Fourier transform of the spike-triggered average waveform by the Fourier transform of the autocorrelogram of the presynaptic VB neuron, followed by an inverse Fourier transform (Brillinger et al. 1976; Eggermont et al. 1993).

6 This idealized kernel can be thought of as the waveform that might result from a single, temporally isolated spike in a single VB thalamocortical neuron that was uncorrelated with spikes of any other neurons that project to the recording site.

7 This is based on an estimated barrel diameter and thickness of 400 µm (McMullen et al. 1994a) and a density of parvalbumen-positive GABAergic interneurons of 7,100/mm3 (McMullen et al. 1994b).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 23 September 1999; accepted in final form 17 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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