These findings indicate that IB cells receive less GABAA-mediated inhibitory input and …

• Abstract
• Introduction
• Methods
• Results
• Discussion
• Miscellaneous
• References
• Table 1
• Figures

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 GABA_A input(Fig. 5Ac, 60 mV; 5Bc, 64 mV). A number of attempts were madeto confirm that this PSP was mediated by GABA_A, 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 theGABA_A, 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 GABA_A in RS cells was a long, slow hyperpolarization,which was likely mediated by GABA_B receptors (Fig. 5, Ab and Bb).This was confirmed through several experiments in which this hyperpolarizationwas blocked by saclofen (data not shown). The GABA_B 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, GABA_A was not associated with a GABA_B IPSP. Onlyone IB cell IPSP appeared to have a GABA_B 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 GABA_A and GABA_B 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 GABA_A block with TS-TM calix[4]arene and TS calix[4]arene

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 GABA_A, 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 GABA_A inputs were significantly steeperthan those of IB cell GABA_A inputs (P < 0.05).

To investigate this result further, we sought to pharmacologically isolate excitatory synaptic responses. Use of a bath-appliedGABA_A blocker was undesirable for two reasons: first, bath-appliedGABA_A 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 GABA_A current intracellularlythrough addition of TS-TM calix[4]arene and TS calix[4]arene 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 GABA_A 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 GABA_A 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 GABA_A and GABA_B responsesin RS cells (Figs. 8A and 9A). The GABA_A 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 beforeGABA_A blockade, but the EPSP now caused one or more spikes insteadof being shunted by inhibitory input. The GABA_B IPSP remains stableboth in amplitude and duration (Figs. 8B and 9C), indicating thatthe GABAergic input is still present, but the GABA_A componentis blocked. It was also sometimes possible to observe intermediatestages of the GABA_A blockade (Fig. 9B). The unveiling of a considerablesuprathreshold excitatory event was unexpected, because excitationwas not always prominent in the original synaptic responses. OnGABA_A blockade, however, the excitatory input was always quitelarge, consistently causing at least one, and up to four actionpotentials.

IB cells that lacked an apparent GABA_A 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 GABA_A 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.