- Anatomy, Physiology, and Synaptic Responses of Rat Layer V Auditory Cortical Cells and Effects of Intracellular GABAA Blockade
This report represents the first systematic study of the anatomy and physiology of single cells in layer V of auditory cortex.Although part of this work replicates experiments performed elsewherein cerebral cortex, it is necessary to establish that auditorycortex is organized similarly to other sensory cortices beforeother properties can be explored. The role of inhibition in shapingthe synaptic responses of IB and RS cells has been addressed ina number of ways, and it is the major finding of thisreport.
Intrinsic and anatomic properties
As reported in other cortical areas, IB cells have large cell bodies and long, thick apical dendrites that branch extensivelyin layer I. Their axons project into the subcortical white matterand arborize locally in the infragranular cortical layers. RScells have smaller cell bodies and a thinner apical dendrite thatseldom extends to layer I. Their axons also project toward thewhite matter and arborize locally in supragranular cortex. Wheninjected with current, IB cells fire a characteristic burst ofaction potentials, followed by either additional bursts or singlespikes. RS cells fire single spikes with a variable degree ofadaptation. These findings suggest that in layer V, primary sensorycortices share organizational features across sensorymodalities.
Thalamocortical input to layer V
Stimulation of the white matter in coronal slices and thalamocortical inputs in horizontal slices produced consistent synapticresponses in both RS and IB cells. When stimulating a fiber tract,there is always the possibility that cortical projection neuronsare antidromically activated concurrent with stimulation of thalamocorticalfibers. At low and moderate stimulation strengths, it was exceedinglyrare to antidromically activate a recorded cell in layer V, orin cells recorded in other cortical layers, although synapticresponses were always observable. This is likely because thalamocorticalfibers are thicker than both corticocortical and corticothalamicfibers (Katz 1987; McGuire et al. 1984), and their threshold foractivation is lower (Bullier and Henry 1979; Ferster 1990; Fersterand Lindstrom 1983, 1985). Because antidromic spikes were rarelyobserved, and because low to moderate stimuli were generally used,we concluded that the synaptic responses observed are primarilythe result of thalamocortical fiberactivation.
RS cells received excitation followed by GABAA and GABAB IPSPs at latencies indicative of di- or trisynaptic inputs. TheseRS cell synaptic inputs may have been mediated by cells in layerIII/IV receiving direct, suprathreshold thalamic input (Agmonand Connors 1992; Hirsch 1995). Suprathreshold responses wererare unless the cell was depolarized, suggesting that RS cellsrequire concurrent inputs to reach spike threshold. IB cells receivedexcitatory synaptic input either at short latencies, seen onlyin horizontal slices and suggesting a monosynaptic input, or atlonger latencies suggesting a di- or trisynaptic nature. Thissuggests that thalamocortical input to IB cells can be separatedinto two channels. The first channel is a fast, probably monosynapticsuprathreshold input. This direct thalamocortical input couldarrive on the apical dendrite of the IB cell in layer IV (Kurodaet al. 1995, 1996, 1998). The second channel is a longer latencymulticomponent EPSP that may represent input from another IB cell.The EPSP components had the same interevent interval and timecourse as an IB cell burst, and the multiple-component EPSP wasonly seen at longer latencies, supporting this speculation. Largelayer V cells, morphologically identical to IB cells, are synapticallyconnected (Gabbott et al. 1987; Markram 1997), also supportingthisidea.
Less than half of IB cells received any identifiable inhibitory input, and stimulation usually caused an action potentialor burst, even from rest. The difference in the amount of inhibitoryinput to RS and IB cells was the most striking finding among thesynaptic data. IB cells received inhibition less often than RScells and lacked a GABAB IPSP. This lack of strong inhibitioncontributes to the increased ability of IB cells to spike in responseto synaptic input in vitro, and may have this effect in the intactsystem.
Responses of RS and IB cells to stimulation of their synaptic inputs are similar to those observed in other sensory cortices(Baranyi et al. 1993; Chagnac-Amitai and Connors 1989; Nunez etal. 1993). One obvious difference exists between the present findingsand a previous study (Agmon and Connors 1992). In that study,most IB cells observed (5 of 7) in somatosensory cortex did notappear to receive any obvious thalamocortical input, which isat variance with the current report. The simplest explanationfor this discrepancy is that the stimulation methods used, inwhich thalamic areas were stimulated, activated a smaller proportionof the total thalamocortical input than the fiber tract stimulationthat was used in the current report. In rat motor cortex (Castro-Alemancosand Connors 1996) IB cells appear to receive strong inhibitionthat can be activated through stimulation of their thalamic inputs.This suggests that pyramidal cells may have different inputs basedon the cortical area in which they are found. Differences in inhibitionbetween RS and IB cells have also been observed (Chagnac-Amitaiand Connors 1989; Nicoll et al. 1996). Synaptic responses in auditorycortex have been described previously (Cox et al. 1992; Metherateand Ashe 1991, 1993, 1995); however, laminar locations were seldomreported.
IB cells may be well suited to generate synchronized bursts of activity given their relative lack of inhibition (Chagnac-Amitaiand Connors 1989). Interconnections exist between IB cells (Markram1997) and between IB and RS cells (Gil and Amitai 1996), a necessaryfeature for generating this type of synchronous activity. In addition,some IB cell interburst intervals match the frequency of corticaloscillations observed both in vivo and in vitro, and layer V isboth necessary and sufficient to produce synchronous corticalactivity (Silva et al. 1991). Our synaptic stimuli revealed inputsto IB cells that matched an IB cell burst in both interevent intervaland overall duration, suggesting that at least a portion of theinterconnections between IB cells is retained and can be activatedinslices.
Intracellular block of GABAA inhibition
Intracellular GABAA blockade demonstrated that inhibitory current strength differs between RS and IB cells and confirmed manyearlier experiments in which inhibition was assessed indirectly.Intracellular chloride blockers also revealed a large excitatoryevent in RS cells, which is not seen under normal conditions.RS cells, on GABAA blockade, produced a series of action potentials(unlike the IB cell burst, in pattern and frequency), even whenthe synaptic response formerly contained little discernable excitatorycomponent. The responses of IB cells were less dramatic, oftenproducing an additional spike where an IPSP was formerly seen,or showing no effect in IB cells in which no GABAA wasobserved.
These data indicate that the total excitation reaching RS cells is at least as robust as that seen in IB cells. Two questionsare whether the inhibitory inputs are activated in vivo to thedegree that they are in vitro, and whether they are activatedconcurrent with the excitation. Intracellular recordings fromcat auditory cortex during auditory stimulation in vivo revealtwo response types in layers V and VI (Volkov and Galazjuk 1991).Phasic responders, which resemble RS cells in their intrinsicphysiology, are excited at tone onset, and thereafter are activelyinhibited. Tonic responders, which resemble IB cells physiologically,fired a train of spikes or bursts throughout the tone stimulus.Tonic cells seldom showed inhibition and were more broadly frequency-tunedthan phasic neurons. This suggests that the strong, thalamocorticallydriven inhibition reaching RS cells forms an inhibitory "surround,"sharpening RS cell responses. This idea is well established invisual cortex. There, noncorticotectal layer V pyramidal neurons(RS cells) have small receptive fields and narrow orientationand directional selectivity (Finlay et al. 1976; Swadlow 1988).Large layer V corticotectal cells, identified as IB cells (Kasperet al. 1994a; Rumberger et al. 1998), have broader receptive fieldsand selectivity (Finlay et al. 1976; Swadlow 1988), suggestingthat IB cells lack the strong inhibitory input that sharpens RScell responses to sensory stimuli. The in vivo data fit well withthe present findings and indicate that tuning in IB and RS neuronsmay be shaped byinhibition.
Comparison to in vivo auditory cortical studies
Extracellular studies of auditory cortex reveal neurons sensitive to many aspects of sound stimuli. Some studies note activitydescribed as "bursts" (Evans and Whitfield 1964). Extracellularresponses to vocalizations in the squirrel monkey (Glass and Wollberg1979; Wollberg and Newman 1972) also display spike patterns reminiscentof IB cell bursts, which recur consistently in response to oneportion of the call. Although it is impossible to say that theburstlike behavior described above originates from IB cells, itsuggests that bursts may be physiologically relevant in the intactsystem. As previously suggested (Lisman 1997), bursting cellsmay serve as event detectors. Bursts may also have a higher signal-to-noiseratio and could sharpen frequency tuning (Eggermont and Smith1996). Clearly more experimentation is needed to characterizethe response properties of these bursting cells and to identifythem directly with IB cells reported invitro.
Possible roles of feedback projections from layer V
Anatomic evidence suggests that IB cells are the source of layer V input to the MGB (Winer 1992), inferior colliculus (IC)(Games and Winer 1988; Moriizumi and Hattori 1991), and cochlearnucleus (Weedman and Ryugo 1996a,b). Cells anatomically similarto RS cells project to other cortical areas (Games and Winer 1988),and to the putamen (Ojima et al. 1992).
One unique feature of layer V cells in sensory cortex, identified anatomically as IB cells, is the very large (often >5 µm)synaptic contacts they make in secondary thalamic areas (Bourassaand Deschenes 1995; Hoogland et al. 1991; Roullier and Welker1991). These contacts may constitute a "driving input" (Guillery1995; Miller 1996; Sherman and Guillery 1996), in contrast tothe layer VI corticothalamic feedback which is "modulatory." Inthe posterior complex (Po) of somatosensory thalamus, which receiveslarge layer V synaptic contacts, cortical inactivation made cellsunresponsive to sensory stimuli (Diamond et al. 1992). This implicateslayer V as providing necessary sensory information to secondarythalamic areas and supports the idea that layer V projectionsare "driving" inputs. The IC receives its cortical input exclusivelyfrom layer V. Activating auditory cortex enhances IC cell responsesat the peaks of their tuning curves and inhibits responses off-peak(Sun et al. 1996; Yan and Suga 1996). Putative corticocollicularsynaptic contacts are small (Saldana et al. 1996), supportingthis apparent modulatory role in the IC, although synchronizedlayer V activity may be capable of driving ICneurons.
The combined anatomic and physiological evidence indicates very different roles for IB and RS cells in cortical and subcorticalcircuitry. Most RS cells may participate in a feed-forward pathwayfrom primary to secondary and contralateral auditory cortices.IB cells, in contrast, make up the majority of layer V's inputto subcortical targets such as the MGB and IC and may providedriving inputs in secondary thalamic areas. This creates an alternativecorticocortical pathway, through secondary thalamus (Guillery1995; Sherman and Guillery 1996). Corticothalamocortical synapticinput may be stronger, and therefore more effective, than directcorticocortical projections, as supported by in vivo data andRS cell thalamocortical responses in vitro. Based on evidencefrom our experiments, RS cells are strongly inhibited and mayprovide less robust, but perhaps more specific, information aboutsensory stimuli to their synaptic targets. In contrast, IB cellsreceive less inhibition and are capable of providing a robustinput to anytarget.
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