Department of Anatomy and Neuroscience Training Program, University of Wisconsin Medical School, Madison, Wisconsin 53706
The Journal of Neurophysiology Vol. 83 No. 5 May 2000, pp. 2626-2638.
Hefti, Brenda J. and
Philip H. Smith.
Anatomy, Physiology, and Synaptic Responses of Rat Layer V
Auditory Cortical Cells and Effects of Intracellular GABAAJ. Neurophysiol. 83: 2626-2638, 2000.
The varied extracortical targets of layer V
make it an important site for cortical processing and output, which may
be regulatedby differences in the pyramidal neurons found there. Two
populationsof projection neurons, regular spiking (RS) and intrinsic
bursting(IB), have been identified in layer V of some sensory
cortices,and differences in their inhibitory inputs have been
indirectlydemonstrated. In this report, IB and RS cells were
identifiedin rat auditory cortical slices, and differences in
thalamocorticalinhibition reaching RS and IB cells were demonstrated
directlyusing intracellular GABAA blockers.
Thalamocortical synaptic inputto RS cells was always a combination of
excitation and both GABAAand GABAB inhibition.
Stimulation seldom triggered a suprathresholdresponse. IB cell
synaptic responses were mostly excitatory, andstimulation usually
triggered action potentials. This apparentdifference was confirmed
directly using intracellular chloridechannel blockers. Before
intracellular diffusion, synaptic responseswere stable and similar to
control conditions. Subsequently, GABAAwas blocked,
revealing a cell's total excitatory input. On GABAAblockade, RS cells responded to synaptic stimulation with large,suprathreshold excitatory events, indicating that excitation,while
always present in these cells, is masked by GABAA. In IBcells that had visible GABAA input, it often masked an
excitatorypostsynaptic potential (EPSP) that could lead to additional
suprathresholdevents. These findings indicate that IB cells receive
less GABAA-mediatedinhibitory input and are able to spike
or burst in response tothalamocortical synaptic stimulation far more
readily than RScells. Such differences may have implications for the
influenceeach cell type exerts on its postsynaptictargets.
Auditory cortex is the last in a series of structures dedicated to the interpretation of auditory input. Many subcorticalauditory nuclei have specialized circuits or synapses that haveno correlates in the other sensory systems. For instance, themedial nucleus of the trapezoid body (MNTB) contains large calycealsynapses specialized for rapid, precise synaptic transmission(Trussell 1997). Recent evidence (Smith and Populin 1999) suggeststhat the thalamic input layers of cat auditory cortex may differfrom those of visual and somatosensory cortices. It is possiblethat rat auditory cortex also contains unique circuits and cellsspecialized for auditory information processing. Alternatively,auditory cortex may process stimuli using circuits similar tothose found elsewhere in cortex. It is therefore important tokeep these possible specializations in mind and study auditorycortex both in terms of its possible auditory functions and asa part of cerebralcortex.
Sensory cortex influences its targets through a topographically organized descending system originating in layers V and VI,and recent work has begun to elucidate the possible physiologicalroles of this system (Guillery 1995; Miller 1996; Sherman andGuillery 1996). Layer V is of particular interest because itscells form part of the projection to the thalamus and the entiretyof the cortical projection to subthalamic nuclei. In addition,layer V, with cells from all cortical layers (barring layer I),participates in callosal and ipsilateral corticocortical circuits.Layer V has several anatomic and physiological pyramidal celltypes and a variety of interneurons (Kawaguchi 1993; Kawaguchiand Kubota 1996). One pyramidal cell type, the intrinsically bursting(IB) cell, is found only in layer V and the deepest region oflayer IV in the rat (Connors et al. 1982, 1988; McCormick et al.1985, somatosensory cortex; Kasper et al. 1994a, visual cortex).The IB cell produces high-frequency bursts of action potentialsand is distinguished by its morphology, which is different fromthat of regular spiking (RS) cells, which also populate layerV (Chagnac-Amitai et al. 1990; Kasper et al. 1994a-c). It is possiblethat the diverse targets of layer V necessitate a variety of anatomicand physiological response types, but the role of these differenttypes within layer V and their effects on postsynaptic targetsare only beginning to be understood (Guillery 1995; Miller 1996;Sherman and Guillery 1996).
In this study we characterized the physiological and anatomic properties of single cells in layer V of primary auditory cortex,their synaptic inputs, and how their responses to these inputsmight modulate their cortical and subcortical targets. This reportuses three techniques to approach these issues. First, by examiningascending synaptic inputs to layer V cells, latencies, patterns,and degrees of excitation and inhibition can be identified. Second,intracellular blockers of GABAA allow confirmation of earlierwork, which studied the role of inhibition indirectly (Chagnac-Amitaiand Connors 1989; Nicoll et al. 1996), as well as further studyof the strength of the inhibitory input and its ability to shapethe thalamocortical synaptic responses of layer V cells. Third,anatomic results can be correlated to physiological data to givea clearer picture of auditory and more general cortical circuitry.Part of this work was published previously in abstract form (Heftiand Smith 1996, 1999).
The methods described here for intracellular sharp microelectrode recording are similar to those described previously (Smith1992). All methods were approved by the University of WisconsinAnimal Care and Use Committee. Animals were maintained in an AmericanAssociations for Accreditation of Laboratory Animal Care (AAALAC)-approvedfacility. Three to 6-wk-old Long-Evans hooded rats were givenan anesthetic overdose of chloral hydrate solution (70 mg/ml ip).When areflexive, rats were perfused transcardially with cold,oxygenated sucrose saline (described at end of paragraph). Thebrain was then exposed dorsally, and cuts in the coronal planewere made halfway through the rostrocaudal extent of the cerebellumand one-third of the way through the rostrocaudal extent of thecerebral cortex. The block of tissue between these two cuts wasremoved and glued either ventral side down (for horizontal sections)or rostral side down (for coronal sections). The tissue was thensubmerged in cold, oxygenated saline, and 400- to 500-µm sectionswere cut through primary auditory cortex (Te1) on a vibratome.To preserve more of the axonal projection from the medial geniculatebody (MGB) to Te1 in horizontal slices, the tissue was blockedsomewhat higher rostrally and laterally with wedges of fixed eggalbumin (Metherate 1999). Sections containing Te1 were placedin a holding chamber containing normal, oxygenated artificialcerebrospinal fluid (ACSF) at room temperature. After equilibratingin the holding chamber for at least 15 min, one slice was transferredto the recording chamber, where it was placed between two piecesof nylon mesh and perfused with normal, oxygenated ACSF at 35°C,which contained the following (in mM): 124 NaCl, 5 KCl, 1.2 KH2PO4,2.4 CaCl2, 1.3 MgSO4, 26 NaHCO3, and 10 glucose. The sucrose salinecontained sucrose in place of NaCl (Aghajanian and Rasmussen 1989).The slice was then allowed to rest a minimum of 45 additionalminutes before recordingbegan.
Bipolar stimulating electrodes were used to activate axons in the white matter in coronal slices, with stimuli that were steppedfrom 10 to 150 V in 10-V increments, and with durations of 100 or 200 µs. In horizontal slices, the internal capsule and externalcapsule were stimulated separately, allowing isolation of thalamocorticalfrom corticocortical inputs (Fig. 1). The space between the pairedelectrode tips was sufficient to span the width of the fiber tractto stimulate the maximum number of inputs possible. Occasionally,stimulation induced antidromic spikes from the recorded cell.If this was observed, the polarity of the electrode was switchedor the stimulating electrode was moved.
Intracellular recordings of responses to injected current and evoked postsynaptic potentials were made with glass microelectrodesof 70-150 M resistance when filled with 2 M potassium acetateand 2% Neurobiotin (Vector Laboratories, Burlingame, CA). Onlycells with resting potentials more negative than 60 mV and overshootingaction potentials were used for statistical analysis and illustration.Intracellular current and voltage records were digitized withcustom software (ICEPAC, L. Haberly, University of Wisconsin).A neuron's membrane potential was calculated by subtracting thecell's recorded voltage from the extracellular DC potential justafter exiting the cell. The input resistance was calculated usingthe slope of the linear portion of the current-voltage plot nearthe cell's resting potential. Voltage was averaged over 100 msduring the last 120 ms of a 300- or 400-ms current pulse. Duringrecording, Neurobiotin was injected into the cell for ~5 min with0.4- to 0.6-nA current pulses. To quantify spike half-widths,the first and fifth spikes were measured. The fifth spike waschosen because it was usually the first or second spike afterthe burst in IB cells, and all cells fired at least five spikesin response to current injection. Measurements were taken at thelowest current injection strength at which the cell fired fivespikes, which was always between 0.1 and 0.5 nA. Synaptic latencieswere measured from the center of the stimulus artifact, whichwas usually a total of 0.5 ms in duration, to the onset of thevoltage deflection. Inhibitory postsynaptic potential (IPSP) latencywas measured from the center of the stimulus artifact to the onsetof the IPSP, which was identified as the onset of the change inthe slope of the voltage deflection that reversed at levels correspondingto a chloride or mixed anion conductance (usually between 50and 70mV).
GABAB was blocked with saclofen (Research Biochemicals International, Natick, MA) in three cells, one IB and two RS. GABAA-activatedchloride channels were blocked intracellularly with 5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxi-calixarene(TS-TM calixarene) and 5,11,17,23-tetrasulfonato-calixarene(TS calixarene), which were generously provided by Dr. AshvaniSingh at the University of Pittsburgh. These compounds were usedat a concentration of 1-5 µM. They were injected into the cellafter control trials were taken using hyperpolarizing square currentpulses (300 ms current pulse every 800 ms). It usually took between20 and 40 min for the chloride blockers to takeeffect.
After recording was complete, the slice was fixed in fresh 4% paraformaldehyde. It was then cryoprotected, and 60-µm frozensections were cut on a freezing microtome and collected in 0.1 M phosphate buffer, pH 7.4. The sections were incubated in avidin-biotin-HRPcomplex (ABC kit, Vector Labs). The following day, they were rinsedin phosphate buffer and incubated with nickel/cobalt-intensifieddiaminobenzidine (DAB) (Adams 1981). The sections were then mounted,counterstained with cresyl violet, andcoverslipped.
Drawings of injected cells were made using a camera lucida attached to a Zeiss microscope. The location of the cell body relativeto the areas of rat cerebral cortex was determined using the atlasof Paxinos and Watson (1986) and studies in which evoked potentialrecordings were used to map the location of primary auditory cortex(Barth and Di 1990, 1991; Di and Barth 1992). Cells were determinedto be within layer V by two means. The first was inspection ofindividual sections, where differences in cell size, density andshape were used to indicate transitions between cortical layers.The second means to determine laminar borders in Te1 was to usepreviously established measures of layer V laminar borders (Gamesand Winer 1988), in which layer V is defined as the region ~51-77%of the distance through Te1 when measured from the pial surface.Only those cells that fell both within primary auditory cortex(Te1) and layer V were used for analysis. Anatomic measurementswere made using a Neurolucida drawing system (MicroBrightField,Colchester, VT). All statistical analyses were done using Minitab(Minitab, State College, PA). Depending on the data set, eithertwo-sided two-sample t-tests or 2 tests wereused.
Physiological types in auditory cortex
When cells were recorded from and labeled in layer V of auditory cortex, the large majority showed two distinct patterns ofaction potential firing (Fig. 2) in response to current pulses.These two patterns have been previously observed in vitro (Agmonand Connors 1989, 1992; Connors et al. 1982, 1988; Kasper et al.1994a; McCormick et al. 1985) and in vivo in somatosensory (Liand Waters 1996), motor (Baranyi et al. 1993; Pockberger 1991),visual (Holt et al. 1996), and association (Nunez et al. 1993)cortices, and existing terminology has been used here. Most cellsshowed a RS pattern, which is characterized by a train of singleaction potentials. In all RS cells, firing begins at a relativelyrapid rate, and spike adaptation occurs within the first 50 msof the current pulse, causing spike frequency to decrease. In22 of 67 RS cells, denoted RS1 cells, the cell fires at a constantrate for the remainder of the current pulse (Fig. 2A). When hyperpolarized,these cells seldom have a slow depolarization, sometimes calleda "sag," and they have a very small or absent rebound depolarizationfollowing the current pulse. A useful way to illustrate firingpatterns is to plot spike number against time (Fig. 2A, inset)(Agmon and Connors 1992). Linear portions of the plot representa constant firing rate, whereas curved portions illustrate changesin spike rate. RS1 cells are characterized by a plot that is initiallycurved and then becomes linear for the majority of the currentinjection. The remainder (45 of 67) of RS cells were called RS2cells. In RS2 cells, spike rate adaptation continues, and thecell's firing rate slows throughout the current pulse (Fig. 2B).In some cases, action potential firing slows until the cell ceasesto spike before the current pulse has ended. When hyperpolarized,these cells always showed a slight sag at moderate to large currentinjection strengths, and they always had a rebound depolarizationafter the current pulse. The RS2 cell spike number versus timeplot has no linear portion (Fig. 2B, inset). Although these characteristicdifferences between RS1 and RS2 cells are observable in voltagetraces, a satisfactory means to quantify these differences couldnot be developed. This is most likely because the population ofRS2 cells displayed a range of degrees of spike frequency adaptation,and the classifications RS1 and RS2 likely represent oppositeends of a continuum. There were also no significant differencesbetween RS1 and RS2 cells in any other intrinsic, synaptic, oranatomic properties that can be analyzed in the slice preparation.These types will therefore be treated as one group, denoted RS,for the remainder of this report.
The other type of pyramidal cell observed in layer V of rat auditory cortex is the IB cell. This cell type fires bursts ofthree to five action potentials that ride on a slow depolarizationat low current injection strengths. At higher current strengths,IB cells fire one such burst at the onset of a current pulse followedby a long hyperpolarization, and then single spikes at a regularrate for the remainder of the current pulse (Fig. 2C). When hyperpolarized,these cells often had a sag, but their rebound depolarizationwas either small or absent. This cell's spike number versus timeplot (Fig. 2C, inset) consists of two separate linear portionsof different slopes. Spike frequency within a burst, which averaged180-200 Hz, was constant across all current injection strengthsin an individual cell, and was similar between cells. Spike amplitudedecrement was also a consistent feature of the intrinsic burst.The response pattern, burst frequency, and spike decrement observedhere are similar to other reports of IB cells (Agmon and Connors1989; Connors et al. 1982, 1988; Kasper et al. 1994a; McCormicket al. 1985).
The differing intrinsic properties of RS and IB cells are distinguishable on inspection and can be quantified in a numberof ways (Table 1). IB cell input resistance was significantlylower than that of RS cells. Spike half-widths were also differentbetween cell types. First and fifth spike half-width were bothsignificantly narrower in IB cells than in RS cells. There wasno significant increase in spike half-width in IB cells betweentheir first and fifth spikes, but RS cell spikes did become wider.There was no difference in resting membrane potential betweenIB and RS cells.
Anatomic types in auditory cortex
All recorded cells were injected with Neurobiotin and processed for anatomy to study cell morphology. Ten RS cells and 10 IB cells were selected for anatomic analysis. To minimize samplingerror and bias, cells from both coronal and horizontal sliceswere used, they were selected evenly across experimental dates,and no two cells were selected from the same experiment. Qualitatively,the anatomic appearance of RS cells was strikingly different fromthat of IB cells (Figs. 3 and 4). These differences have beennoted previously in other sensory cortices (Chagnac-Amitai etal. 1990; somatosensory and visual cortex; Kasper et al. 1994a-c;visual cortex). The IB cell apical dendrite was very thick andalways extended to layer I, where it branched profusely, and ithad many dendritic branches in other layers as well. RS cell apicaldendrites were shorter and thinner and had fewer secondary branches(Fig. 3). Quantitatively (Table 1), the IB cell soma was muchlarger than that of RS cells, and the apical dendrite of IB cellswas longer. The apical dendrite of IB cells was also consistentlythicker than that of RS cells when measured at 50, 200, and 400 µm from the soma. Analysis of the 200- and 400-µm data were complicatedby the fact that the RS cell apical dendrite sometimes split intomultiple branches, all of which extended toward layer I. Two differentmeasurements were used to compare them with IB cell apical dendrites.In one, only the thickest branch of each RS cell apical dendritewas measured. In the second, all branches of the apical dendritethat continued toward layer I were added together for an individualRS cell and used as a single measure of apical dendritic width.In both cases, the IB cell dendrite was significantly thickerat all distances.
The number of secondary branches in layers II, III, and IV emerging directly from the apical dendrite also differed betweencell types, with IB cells having more branch points in each layer.There was no difference in number of branches in layer V, mostlikely because of wide variations in soma location within layerV. The distance from the soma to the layer V/IV border in thesampled cells ranged from 10 to 200 µm. All branch points werecounted in layer I, and IB cells had significantly more branchpoints in this layer aswell.
These same 20 cells (10 IB and 10 RS) were used for analysis of local axonal arborizations within primary auditory cortex.Results were similar to those seen elsewhere (Mitani et al. 1985;cat auditory cortex; Chagnac-Amitai et al. 1990; Gabbot et al.1987; Ojima et al. 1992). RS cells had extensive axonal arborizationsthat were often concentrated in the supragranular layers of cortex(8 of 10 cells; Fig. 4, B). In two cells, axon collaterals wereconcentrated in layer V and the subgranular layers (Fig. 4, A).Their main axon always extended toward the subcortical white matter,and it was possible in most cases to trace it into the fiber tract(Fig. 4, A and B). The local axons were very thin, and left boutonsen passant or at the ends of small stalks. IB cells had fewerlocal collaterals, and unlike most RS cells, their axonal projectionswere concentrated in layers V and VI (Fig. 4, C and D). Theirmain axon could also be followed into the subcortical white matter,and locally projecting axons were thin and left boutons en passantor at the ends of smallstalks.
Synaptic responses of RS and IB cells
The plane of section determined which fiber tracts were stimulated in each experiment. In coronal slices, the white matterwas stimulated, activating both thalamocortical (TC) and corticocortical(CC) inputs to Te1 (Fig. 1A). In horizontal slices, these twopathways were separate, and it was possible to stimulate separatelyeither the TC inputs, in the internal capsule, or the CC inputs,in the external capsule (Fig. 1B). Stimulation of the white matteror internal capsule produced similar synaptic responses in RScells (Table 1). RS cells typically responded first with an excitatorypostsynaptic potential (EPSP). The EPSP latency was relativelyconstant (jitter <0.2 ms) in an individual cell, and ranged from1.5 to 4.0 ms across the RS population. The shortest observedsynaptic latencies in this preparation were between 0.5 and 1.0 ms (in IB cells), so it is likely that most RS cell inputs weredisynaptic. In a minority of RS cells sampled, this EPSP becamesuprathreshold at high shock strengths (Fig. 5Ba). Most (36 of56) RS cells could be induced to fire action potentials if theywere also depolarized from their resting potential, includingall RS cells that also fired an action potential at rest. Some(20 of 56) RS cells never fired an action potential in responseto TC or white matter stimulation (Fig. 5A). The cells that neverspiked received strong inhibition that presumably prevented themembrane voltage from reaching spike threshold.
Following the EPSP in most RS cells was an IPSP, with a latency of 2.0-5.0 ms. The IPSP was visible as a depolarizing PSPand not readily distinguishable from the EPSP at rest, becauseits reversal potential was more depolarized than the cells' restingpotential. Polarizing the cell around its resting potential withcurrent injection during synaptic stimulation revealed a reversalpotential that was consistent with a chloride-mediated GABAA input(Fig. 5Ac, 60 mV; 5Bc, 64 mV). A number of attempts were madeto confirm that this PSP was mediated by GABAA, but the additionof bicuculline to the bath caused global depolarization and uncontrolledspontaneous activity, presumably because of tonic inhibition activein control conditions, making it impossible to record usable data.In many (36 of 56) RS cells, another depolarizing potential couldbe seen following the IPSP (Fig. 5Ac). This second EPSP only occurredwhen the cell also received inhibition, and it is unclear whetherit was a continuation of the initial EPSP, interrupted by theGABAA, or a different input. This second depolarization generallydid not trigger an action potential either from resting membranepotential or when the membrane was depolarized. Always associatedwith the GABAA in RS cells was a long, slow hyperpolarization,which was likely mediated by GABAB receptors (Fig. 5, Ab and Bb).This was confirmed through several experiments in which this hyperpolarizationwas blocked by saclofen (data not shown). The GABAB IPSP couldbe quite large, causing a hyperpolarization of up to 7 mV, andlasting from 300 to 650 ms.
IB cells responded to stimulation of the white matter or internal capsule (in which there are ascending thalamocortical axons,but not corticocortical axons) with an EPSP of 1.5-3.0 ms latency.In a few cases (8 of 36), the synaptic response was suprathresholdexcept at the very lowest levels of synaptic stimulation (Fig.6B). Many IB cell EPSPs (18 of 36) contained two or three separatecomponents, and unlike in RS cells, the EPSPs did not necessarilyoccur in the presence of an identifiable IPSP (Fig. 6A). In fact,less than half of IB cells received any apparent inhibitory input,which was significantly less often than RS cells. In addition,unlike RS cells, GABAA was not associated with a GABAB IPSP. Onlyone IB cell IPSP appeared to have a GABAB component, and it wassmall (<1 mV) and relatively short (225 ms). In a small numberof IB cells, internal capsule (TC) stimulation in horizontal slicesproduced synaptic responses of very short latency (0.5-1.0 ms,n = 3 of 10), shorter than any observed during white matter stimulation.This input was always in the form of a single EPSP followed closelyby inhibition. The short-latency EPSP and IPSP were both significantlyfaster than those observed during white matter stimulation (P = 0.01). Of the IB cells recorded in this sample, only one seemedto receive both the fast and the slow TC input (not shown). Itis certain that portions of the synaptic input to a given cellare missing in any brain slice preparation, so it is possiblethat a larger number of cells receive both short- and long-latencyexcitatory input from the thalamus.
No significant differences in PSP latency or amplitude were found between IB and RS cells. Differences between IB and RS cellsynaptic responses were found when their more general responseproperties were compared (Table 1). IB cells were significantlymore likely than RS cells to spike or burst in response to synapticstimulation both at rest and when depolarized. This could be causedby differences in the amount of inhibition each cell type receivesoverall. IB cells received both GABAA and GABAB IPSPs significantlyless often than RS cells. The IB cell's greater ability to fireaction potentials in response to thalamocortical stimulation andtheir relative lack of inhibition compared with RS cells werethe major findings among the synapticdata.
Intracellular GABAA block with TS-TM calixarene and TS calixarene
We have presented data suggesting that RS cells receive greater inhibition in response to thalamocortical and white matterstimulation than do IB cells. One way to roughly assess the strengthof an input is to plot the voltage change it causes versus thecurrent injected into the cell to hold it at a given voltage.The reversal potential of the input is, by definition, at zeroon the y-axis. As a cell's membrane potential is moved fartheraway from this zero point, a rough measure of the strength ofthis input is how much it is able to change the cell's membranepotential toward its reversal potential. A strong input will causea change in the membrane potential that is approximately equalto the distance between the cell's membrane potential and thereversal potential of the input. Such inputs often cause "pointreversals" such as that seen in Fig. 5Ac. Weaker inputs only changethe membrane potential a portion of the distance between the cell'smembrane potential and the reversal potential of the cell, asin Fig. 5Bc. The strength of the input in RS cells versus IB cellshas been illustrated in a plot of current injected versus voltagechange due to GABAA, in which a steeper slope represents a strongerinput (Fig. 7). Only two of each cell type are shown for clarity,but five cells of each type were measured in this way, and theslopes of the RS cell GABAA inputs were significantly steeperthan those of IB cell GABAA inputs (P < 0.05).
To investigate this result further, we sought to pharmacologically isolate excitatory synaptic responses. Use of a bath-appliedGABAA blocker was undesirable for two reasons: first, bath-appliedGABAA blockers at concentrations that totally block that inhibitioncause prolonged epileptiform activity in cortical slices. Second,many observed EPSPs, and all of the IPSPs, are probably di- ortrisynaptic. Bath-applied blockers would interfere with thesecircuits before synaptic responses are recorded at the layer Vcell, confounding data interpretation. It is possible to blockthe chloride channels that mediate the GABAA current intracellularlythrough addition of TS-TM calixarene and TS calixarene tothe recording electrode. These drugs were developed for use incolonic and other tissue for blockade of outwardly rectifyingchloride channels (Venglarik et al. 1994), and they have alsobeen shown to block GABAA receptor channels in visual cortex (Dudekand Friedlander 1996a). Because these compounds take ~30 min todiffuse into a cell, it is possible to record its synaptic responsesboth before and after the inhibitory chloride channels have beenblocked. Another method has also been used to block GABAA intracellularly(Nelson et al. 1994), but this method, in which cesium is presentin the intracellular electrode, has significant effects on thecell's resting membrane potential, input resistance, action potentialwidths, and level of spontaneous activity. These cellular changeshave unknown effects on a cell's responses to synapticstimuli.
As in earlier reports using these drugs (Dudek and Friedlander 1996a,b), no significant changes were observed in any individualcell's resting potential, action potential widths, or input resistance,although cells occasionally displayed spontaneous EPSPs due toan unknown mechanism (data not shown). RS and IB cells had differentresponses to chloride channel block, but this seemed to be correlatedto the apparent strength of the inhibition that was visible atthe soma, rather than to cell type. Evoked synaptic responsesappeared normal and generated robust GABAA and GABAB responsesin RS cells (Figs. 8A and 9A). The GABAA response was blockedafter ~20-60 min of recording, leaving a large excitatory synapticresponse (n = 5; Figs. 8B and 9C). The onset of the excitatoryresponse had the same synaptic latency as was observed beforeGABAA blockade, but the EPSP now caused one or more spikes insteadof being shunted by inhibitory input. The GABAB IPSP remains stableboth in amplitude and duration (Figs. 8B and 9C), indicating thatthe GABAergic input is still present, but the GABAA componentis blocked. It was also sometimes possible to observe intermediatestages of the GABAA blockade (Fig. 9B). The unveiling of a considerablesuprathreshold excitatory event was unexpected, because excitationwas not always prominent in the original synaptic responses. OnGABAA blockade, however, the excitatory input was always quitelarge, consistently causing at least one, and up to four actionpotentials.
IB cells that lacked an apparent GABAA input were unaffected by the addition of the chloride channel blocking drugs (n = 4;Fig. 10A), confirming that recordings of synaptic responses arestable for long periods of time, either in the presence or absenceof these compounds. In those IB cells whose evoked synaptic responsescontained a GABAA component (n = 3; Fig. 10B), it was blocked overa time course similar to that in RS cells. Because IB cells seldomhave robust inhibition, the effects of the GABA channel-blockingdrugs were less dramatic than was seen in RS cells. These cellsoften simply produced another action potential where the inhibitionhad been (Fig. 10Bb), without the dramatic changes in synapticresponse amplitude and shape often seen in the RS cells.
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.
We thank I. Sigglekow, J. Meister, and J. Ekleberry for expert histological processing, Dr. Ashvani Singh for generously providing TS and TS-TM calixarene and helpful comments on its use, and E. Bartlett and M. Banks for discussion of the manuscript and continuing experimental support.
This work was supported by National Institute on Deafness and Other Communication Disorders Grants DC-01999 and DC-00256 and funds provided by a grant to the University of Wisconsin Medical School from the Howard Hughes Medical Institute Research Resources Program for Medical Schools.
Address for reprint requests: P. Smith, Dept. of Anatomy, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706.
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 15 November 1999; accepted in final form 26 January 2000.
|Table 1. Comparisons of IB and RS cell anatomy, intrinsic physiological properties, and synaptic responses|
|Vrest, mV||66.2 ± 4.0||66.4 ± 4.0|
|Rin, M||49.2 ± 14.7||30.7 ± 8.6*|
|First spike half-width, ms||0.93 ± 0.25||0.72 ± 0.14*|
|Fifth spike half-width, ms||1.23 ± 0.46||0.76 ± 0.15*|
|EPSP latency, ms||2.8 ± 1.6||2.5 ± 1.6|
|GABAA present||50 of 56||16 of 36|
|GABAB present||50 of 56||1 of 36|
|IPSP latency, ms||4.4 ± 1.7§||3.5 ± 1.1*§|
|Fast IPSP (GABAA) reversal, mV||60.7 ± 3.6||62.1 ± 5.0|
|Suprathreshold at rest||16 of 56||27 of 36|
|Soma size, µm2||145 ± 21||212 ± 22*|
|Apical dendrite diameter|
|50 µm||2.2 ± 0.4||3.8 ± 0.7*|
|200 µm||2.0 ± 1.3||3.3 ± 0.6*|
|400 µm||0.8 ± 0.4||2.7 ± 0.4*|
|Branches from apical dendrite|
|Layer V||4.8 ± 2.6||7.9 ± 4.8|
|Layer IV||3.4 ± 1.8||10.0 ± 3.3*|
|Layer III||0.8 ± 0.8||5.7 ± 1.9*|
|Layer II||0.1 ± 0.3||0.8 ± 0.9*|
|Total branches in layer I||0.6 ± 1.3||13.1 ± 3.4*|
|Values are means ± SD. Number of RS neurons is 67 and IB neurons is 39. RS, regular spiking; IB, intrinsic bursting; EPSP, excitatory postsynaptic potential; IPSP, inhibitory postsynaptic potential. * Statistically significant difference of at least P < 0.05 using a 2 sample t-test when comparing the RS cell value to the IB cell value. Statistically significant (P < 0.05) for the comparison of RS cell 1st and 5th spikes. Statistical significance of at least P < 0.05 using a 2 test when comparing the RS to the IB cell numbers. § EPSP latencies were significantly shorter (P < 0.01) than IPSP latencies in both IB and RS cells.|
Figure 1 Drawings of slice preparations. A: coronal hemisection illustrating thalamic landmarks and white matter stimulation site (stim). All recordings made in area Te1, primary auditory cortex. B: semihorizontal section illustrating thalamic and midbrain landmarks and internal capsule stimulation site (stim). The tissue is blocked higher rostrally and laterally to give this approximate plane of section. Scale bar is 1 mm. APT, anterior pretectal nucleus; CG, central gray; CPu, caudate putamen; Ent, entorhinal cortex; Hi, hippocampus; IC, inferior colliculus; MD, medial dorsal nucleus; Po, posterior thalamic nucleus; Oc2L, occipital cortex, area 2, lateral; Par1, parietal cortex, area 1; Par2, parietal cortex, area 2; SN, substantia nigra; Te1, primary auditory cortex; Te3, secondary auditory cortex; VP, ventral posterior thalamic nucleus; ec, external capsule; ic, internal capsule; ml, medial lemniscus.
Figure 2 Intrinsic properties of layer V pyramidal neurons. A: intrinsic properties of a regular spiking type 1 (RS1) cell in response to current injection. Notice early spike frequency adaptation (right panel) followed by a sustained firing rate. Left traces are responses to ±0.3 nA, and right are responses to ±0.6 nA of current. B: a regular spiking type 2 (RS2) cell's intrinsic properties. This cell never maintains a sustained firing rate, and its firing rate slows at higher current injection strengths until it ceases to fire. Left: ±0.2 nA. Right: ±0.6 nA. C: intrinsic properties of an intrinsically bursting (IB) cell. This cell type fires a burst of action potentials that ride on a slower depolarization at the onset of a current pulse and then maintains a steady single spike firing rate. Left: ±0.2 nA. Right: ±0.6 nA. Insets: graphic representations of each cell's firing patterns at low () and higher () current levels, illustrating changes in interspike interval. Scale bars at bottom right apply to all voltage traces.
Figure 3 Camera lucida drawings of IB and RS cell somata and dendrites. Examples of the 2 distinct morphologies displayed by IB and RS physiological types. Notice the significantly larger soma and apical dendrite of the IB cells, as well as the characteristic apical dendritic "tuft," which arborizes extensively in layer I. Axonal arborizations have been eliminated for clarity.
Figure 4 Camera lucida drawings of IB and RS cell local axonal arborizations. Examples of the 2 distinct patterns of local axonal projections displayed by IB and RS physiological cell types. Soma and primary dendrite initial segments are shown in black, and axons are shown as solid black lines. RS cell (A and B) axons typically arborize in superficial cortical layers (as in B). Occasionally other patterns are seen (A). IB cell (C and D) local axon collaterals can be found in deep cortical layers in all cases. Inset: low-magnification drawings illustrating soma location and local axonal arborizations within Te1. Sections are oriented similarly to coronal section shown in Fig. 1, and at the same approximate rostrocaudal level. All cells are located within the borders of Te1, denoted by dotted lines. Ventral to Te1 is Te3, also delineated by dotted lines. Scale bar represents 1 mm.
Figure 5 RS cell synaptic responses to stimulation of the white matter. Examples of synaptic responses to white matter stimulation of 2 different RS cells. Results of white matter stimulation were indistinguishable from internal capsule stimulation. Aa: synaptic responses (100 and 150 V) to white matter stimulation at resting potential. GABAA is depolarizing here, and not easily distinguishable from the excitatory postsynaptic potential (EPSP), as was typical of RS cells. Ab: same synaptic responses as illustrated in Aa, but shown at a longer time scale to illustrate GABAB. Ac: stimulation of white matter input (100 V) while changing the cell membrane potential around rest to illustrate GABAA reversal, which converges to a single membrane potential. This was typical of RS cells that had strong inhibitory inputs. Horizontal line represents the resting membrane potential of the cell. Voltage scale in Aa applies to all 3 traces. Ba: another example of an RS cell synaptic response, shown at different intensities to illustrate both supra- and subthreshold responses. Action potential is truncated. Bb: subthreshold responses (50 and 100 V) shown at a longer time scale to illustrate emergence of GABAB. Bc: polarization of the membrane potential around rest (100 V) to illustrate GABAA reversal and suprathreshold response when the membrane is depolarized. Action potentials are truncated. Horizontal line represents the resting membrane potential of the cell. Voltage scale in Ba applies to all 3 traces. Resting membrane potentials were 70 mV (Aa-Ac) and 63 mV (Ba-Bc).
Figure 6 IB cell synaptic responses to stimulation of the white matter.
Aa: synaptic response at 2 stimulation strengths to
illustrate the 3 components of the response. There was no
GABAB. Ab: polarization of the membrane
around rest to illustrate consistency of this synaptic response and
illustrate suprathreshold response with depolarization. The horizontal
line represents the resting membrane potential of the cell. Scale bars
between a and b apply to both voltage
traces. Ba: synaptic response at different stimulation
strengths to illustrate transition from subthreshold to single spike to
dual spike response. Bb: stimulation of the white matter
input while polarizing the membrane to different potentials to
illustrate inhibition (after 1st spike) and robustness of synaptic
response. Horizontal line represents the resting membrane potential of
the cell. All action potentials were truncated so that EPSPs are more
visible. Resting membrane potentials were 68 mV
(Aa-Ac) and 64 mV (Ba-Bc).
Figure 7 Comparison of RS and IB cell GABAA inputs. Plot of current injected vs. the voltage change (voltage change due to GABAA minus steady-state voltage, in mV) due to GABAA, which is a rough measure of the strength of the synaptic input. RS cells consistently have plots with a steeper slope, indicating a stronger input. Five RS and 5 IB cells were measured in this way, and the slopes of this input were significantly different between groups (P < 0.05). Illustrated are 2 cells from each group, RS cells with filled square symbols and generally steeper slopes, IB cells with open circular symbols and shallower slopes.
Figure 8 RS cell white matter synaptic response before and during intracellular
GABAA blockade. A: synaptic response of RS
cell before chloride channel blockade at a short time scale
(top), and at a longer time scale
(middle) to illustrate GABAB current.
Bottom: polarization of the membrane around rest during
stimulation to illustrate GABAA synaptic component.
Horizontal line represents the resting membrane potential of the cell.
B: same synaptic response shown after intracellular
diffusion of TS-TM calixarene, which blocks GABAA.
Synaptic latency is unchanged, but excitatory response has become much
larger, producing a series of 3-4 action potentials. The
GABAB component of the inhibition is unchanged.
GABAA appears to be absent on membrane polarization at this
stimulus strength (B, bottom) and at all other stimulus
strengths tested (data not shown). Both voltage and time scales in
A, top and B, top apply to
A, bottom and B, bottom,
and voltage scales in A and B, top
apply to A and B, center. Resting
membrane potentials were 75 mV (A, top, middle), 70
mV (A, bottom), and 68 mV (B, all).
Figure 9 RS cell white matter synaptic response before and at 2 stages during intracellular GABAA blockade. Another example of blockade of inhibitory input, showing the transition between normal (left) and absent (right) inhibitory input. The GABAB component of the IPSP remains present, but becomes partially obscured at right due to prolonged depolarization in response to synaptic stimulation. Polarization around rest (bottom traces) best illustrates gradual blockade of fast inhibition. Horizontal line represents the resting membrane potential of the cell. Action potentials were truncated in the bottom voltage traces in B and C. Scale bars at top and bottom left apply to all traces at top and bottom, respectively. Resting membrane potentials were 70 mV (A, all), 66 mV (B, all), and 67 mV (C, all).
Figure 10 IB cell white matter synaptic responses in the presence of TS-TM calixarene. A: IB cell without apparent inhibition. The synaptic response remains unchanged after >2 h of recording, and after chloride channel blockers are presumed diffused into the cell. Voltage scale at top applies to all 4 traces. Time scale at top applies to both traces in Ab. B: IB cell with inhibition. GABAA is apparent, although partially obscured by the suprathreshold response in the top 2 panels. When blocked, another action potential can be seen in response to identical synaptic stimulation in the bottom 2 panels. Voltage scale at top of B applies to all 4 traces. Time scale at top also applies to Bb, top. Resting membrane potentials were 71 mV (A, all) and 74 mV (B, all).