Figures

- Anatomy, Physiology, and Synaptic Responses of Rat Layer V Auditory Cortical Cells and Effects of Intracellular GABAA Blockade

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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.

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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.

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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.

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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.

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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. GABA_A 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 GABA_B. Ac: stimulation of white matter input (100 V) while changing the cell membrane potential around rest to illustrate GABA_A 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 GABA_B. Bc: polarization of the membrane potential around rest (100 V) to illustrate GABA_A 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).

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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 GABA_B. 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).

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Figure 7   Comparison of RS and IB cell GABA_A inputs. Plot of current injected vs. the voltage change (voltage change due to GABA_A minus steady-state voltage, in mV) due to GABA_A, 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.

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Figure 8   RS cell white matter synaptic response before and during intracellular GABA_A 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 GABA_B current. Bottom: polarization of the membrane around rest during stimulation to illustrate GABA_A synaptic component. Horizontal line represents the resting membrane potential of the cell. B: same synaptic response shown after intracellular diffusion of TS-TM calix[4]arene, which blocks GABA_A. Synaptic latency is unchanged, but excitatory response has become much larger, producing a series of 3-4 action potentials. The GABA_B component of the inhibition is unchanged. GABA_A 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).

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Figure 9   RS cell white matter synaptic response before and at 2 stages during intracellular GABA_A blockade. Another example of blockade of inhibitory input, showing the transition between normal (left) and absent (right) inhibitory input. The GABA_B 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).

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Figure 10   IB cell white matter synaptic responses in the presence of TS-TM calix[4]arene. 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. GABA_A 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).