Intrinsic electrophysiology of neurons in thalamorecipient layers of developing rat auditory cortex

Developmental Brain Research 115 Ž1999. 131–144 www.elsevier.comrlocaterbres

Research report

Intrinsic electrophysiology of neurons in thalamorecipient layers of developing rat auditory cortex Raju Metherate ) , V. Bess Aramakis Department of Neurobiology and BehaÕior and Center for the Neurobiology of Learning and Memory, UniÕersity of California, IrÕine, 2205 Biological Sciences II, IrÕine, CA 92697-4550, USA Accepted 6 April 1999

Abstract During early postnatal life, several critical events contribute to the functional development of rat sensory neocortex. Thalamocortical innervation of sensory cortex is completed during the first postnatal week and extrathalamic innervation develops over the first several weeks. In auditory cortex, acoustic-evoked potentials first occur in week 2 and develop most rapidly over weeks 2–3. Thus, rapid functional maturation of cortical circuits in sensory cortex occurs during the second and third postnatal weeks. The electrophysiological properties of cortical neurons that receive afferent inputs during this time may play an important role in development and function. In this study we examined the intrinsic electrophysiology, including spiking patterns, of neurons in layers IIrIII and IV of auditory cortex during postnatal weeks 2 and 3. Many neurons displayed characteristics consistent with previous descriptions of response classes Žregular spiking, fast spiking, intrinsic bursting.. In addition, we identified two groups, Rectifying and On-spiking neurons, that were characterized by Ži. brief spike trains in response to maintained intracellular depolarizations, and Žii. striking outward rectification upon depolarization. Unusually brief spike trains Ž1–2 spikes. and short spike latencies Ž- 10 ms. further distinguished On-spiking from Rectifying cells. Biocytin labeling demonstrated that On-spiking and Rectifying cells could be either pyramidal or nonpyramidal neurons. The intrinsic physiology of these cell groups may play an important role in auditory cortex function. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Sensory cortex; Onset cell; Intrinsic property; Postnatal development; Thalamocortical; Outward rectification

1. Introduction A convergence of several events during the second and third weeks after birth likely plays an important role in the development of rat sensory cortex. The first postnatal week witnesses the arrival of thalamocortical afferents in layer IV of sensory cortex around postnatal days ŽP. 3–4 w8,29,56,69x. Extrathalamic afferents from the cholinergic basal forebrain that are thought to regulate cortical development w17,55x also begin to innervate the cortex at this time w6,13,36x. By the end of the first week, thalamocortical terminals have reached their adult laminar pattern and density, and cholinergic afferents innervate all cortical layers. Cholinergic terminations continue to mature in terms of laminar patterns and density w6,36x. In the auditory system, functional development proceeds rapidly in postnatal weeks 2 and 3. Acoustic stimuli first elicit immature cochlear potentials on P8–9 w12x and cortical evoked ) Corresponding [email protected]

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potentials on P10–13 w19,41x. The amplitude, latency and complexity of cortical evoked potentials change rapidly during the third week and then mature fully at a slower rate over several weeks w19,41x. Thus, the second and third weeks of postnatal life witness massive synaptogenesis Žsee also Ref. w1x. and rapid functional maturation that likely involves experience-dependent formation of synaptic circuitry. As part of ongoing studies of cortical and cholinergic function during this period w3,18,37,40x, we have examined the intrinsic electrophysiological properties of neurons in auditory cortex with the ultimate goal of understanding how responses to sensory inputs develop. We have focused on neurons in layers IIrIII and IV as they are the main recipients of lemniscal thalamocortical projections w58,67,68x. Intrinsic electrophysiology generally refers to whole-cell electroresponsiveness resulting from the types and distributions of ion channels in the membrane. These properties determine postsynaptic responses, including patterns of spike discharge, to afferent inputs. A widely-used system of classifying cortical neurons by their intrinsic properties

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R. Metherate, V.B. Aramakisr DeÕelopmental Brain Research 115 (1999) 131–144

w10,32x focuses primarily on the patterns and characteristics of spike discharge elicited by depolarizing current pulses delivered via intracellular recording electrodes w2,21,26,46x. Neurons generally are classified as regularspiking ŽRS., fast-spiking ŽFS., and intrinsic-bursting ŽIB.. Additional cell types Že.g., ‘chattering’ cells w15x. and subtypes of existing classes Že.g., RS1–2 w2x. have been described since. In each case, the pattern of spike discharge is thought to reflect a functional contribution of the cell type to cortical circuits. To fully understand how auditory cortex processes sensory information, we must determine the degree to which intrinsic properties contribute to acoustic-evoked responses w38,65x. For example, cortical neurons generally respond transiently to acoustic stimuli, falling silent after discharging only one or a few spikes w7,51x, whereas some neurons in the subcortical auditory system can respond in a more sustained manner w11,49,52x. Explanations for the transient discharge of cortical neurons generally revolve around the strength of intracortical inhibition w7,42,54,66x and the use of anesthesias that depress cortical excitability andror enhance GABAergic inhibition w43,44x. Such factors undoubtedly contribute to neuronal excitability, but intrinsic properties must also contribute to responsiveness, since both synaptic and intrinsic mechanisms shape evoked responses. Here, we continue the task of determining the cellular bases of acoustic responsiveness by examining the intrinsic electrophysiology of neurons in layers IIrIII and IV of developing auditory cortex. 2. Materials and methods Details of the slice preparation and electrophysiological recordings are as published recently w3x. Briefly, Sprague– Dawley rats of either sex and age 8–23 days were anesthetized with barbiturate or halothane and decapitated. Brains were removed into cold artificial cerebrospinal fluid ŽACSF. containing Žin mM.: NaCl 125.0, KCl 2.5, NaHCO 3 25.0, KH 2 PO4 1.25, MgSO4 1.2, CaCl 2 2.0, dextrose 10.0, bubbled with 95% O 2 , 5% CO 2 . Coronal slices Ž300–350 mm. containing auditory cortex were obtained based on landmarks visible in coronal sections w48x and acetylcholinesterase histochemistry Žwhich delineates primary sensory cortex in juvenile rats; see Refs. w3,56x., and placed in a holding chamber containing oxygenated ACSF at room temperature. For recordings, each slice was transferred to a submersion chamber on a fixed-stage microscope ŽZeiss Axioskop. and maintained at 348C. Infrared differential interference contrast ŽIR-DIC. optics enabled visualization of individual neurons and precise placement of recording electrodes w63x. Whole-cell recordings were obtained with patch pipettes Ž4–6 MV . filled with Žin mM.: KMeS0 3 125.0, CaCl 2 0.05, NaCl 0.5, Mg-ATP 2, Na-GTP 0.5, EGTA 0.16, HEPES 10, and 0.3–0.5% biocytin ŽpH 7.3 with KOH, final osmolality 270–280 mosMrkg.. Neural responses

were amplified Ždc-2 kHz, Axoclamp 2B, Axon Instruments., viewed on an oscilloscope and chart recorder, and digitized at 5 kHz for viewing and storage on computer. Series resistance was typically less than ; 15 M V and was fully compensated using the bridge balance. Note that the low resistance patch electrodes did not rectify even upon passage of positive or negative currents much greater than those used here. Membrane potential Ž Vm . was not corrected for junction potential which was estimated to be ; 13 mV Žrange 12–14 mV, n s 3, measured by replacing bath ACSF with pipette filling solution while using a free-flowing KCl reference electrode .. Software ŽAXODATA, AXOGRAPH, Axon Instruments. controlled data acquisition and analysis. Measurements of intrinsic electrophysiology were made within ; 5 min of establishing whole-cell recordings, after Vm stabilization but before significant washout was likely Žobservations repeated after 30–90 min revealed reduced membrane rectification andror reduced spike-frequency adaptation over time for some cells, but not all, presumably due to washout of cellular contents.. Measurements of passive and spike properties were obtained by passing rectangular current pulses ranging approximately from y0.2 to q0.2–0.7 nA in steps of 0.02–0.1 nA Žtypically 0.05 nA.. Apparent input resistance Ž R i . and membrane time constant Žt . at resting Vm were estimated from the initial response Žpeak hyperpolarizing deflection. to a y0.1 nA current pulse. Time constants were estimated by curve fitting Žsingle exponential function.. Spike threshold, height and width were determined from the first spike elicited at a threshold intensity current pulse Žnote that ‘threshold’ in the text below can refer either to spike threshold, in mV, or to the minimum effective current stimulus intensity, in nA, for generating a spike; threshold current intensity was determined typically with 0.05 nA resolution.. Spike height was measured from threshold Žnot resting. Vm and spike width was measured at half height. The latency to spike onset Žat threshold. was determined from the onset of the current step. After each recording, the distance between the soma and the pia was measured using a microscope reticule Žresolution 2.5 mm.. The cell was assigned to a layer by determining its depth relative to a cortical width of 1500 mm Žmean cortical width 1501 " 24 mm, n s 14 slices., and relating the value obtained to published laminar boundaries for rat auditory cortex w57x. Slices with biocytin-filled neurons were placed in 4% paraformaldehyde overnight, then in 0.1 M phosphate buffer ŽPB. for 1–7 days. Slices were washed in PB, incubated in ABC complex ŽVector Labs. for 2–18 h, then rinsed in PB and in Tris buffer. The tissue was then preincubated in 0.02% diaminobenzidine ŽDAB, in Tris with 0.25% nickel ammonium sulfate. for 20 min, then reacted in 0.006% H 2 O 2 q DABrnickel for 4–5 min. Sections were washed in Tris and in PB, then mounted on slides, dried, dehydrated, cleared and coverslipped. Recov-

R. Metherate, V.B. Aramakisr DeÕelopmental Brain Research 115 (1999) 131–144

ered neurons were viewed on a microscope equipped with a drawing tube ŽZeiss Axioskop. and drawn with a 40 = or 100 = oil-immersion objective. All mean values reported are "1 S.E.M. Statistical comparisons ŽStatView software. of passive and spike properties among the main cells groups were performed using ANOVA and Scheffe’s F-post hoc procedures. Comparisons of OS cell characteristics with those of RS and RECT cells Žsee below for definitions of cell types. used one- or two-tailed t-tests, where appropriate.

3. Results The data derive from 233 cells recorded in layers IIrIII and IV. The cells had stable resting Vm more negative than y50 mV Žmean y67 " 0.4 mV. and overshooting action potentials Žamplitude 69 " 0.6 mV measured from spike threshold of y40 " 0.3 mV; note that Vm values are not corrected for junction potential of ca. y13 mV; see Section 2.. Of these, 187 cells could be assigned to one of five categories based on intrinsic electrophysiology. These five categories are the conventional RS, FS, and IB groups, as well as two additional groups, rectifying ŽRECT. and on-spiking ŽOS. cells Ždefined below.. Of the 46 cells that were not assigned to one of these groups, most had characteristics that overlapped two groups Žusually RS and RECT. and could not be placed easily into one group or another. A very few cells had characteristics that did not resemble those of any group or were insufficiently characterized to classify. Data from unclassified cells are not explicitly discussed but contribute to the analysis of agerelated changes in neuronal properties ŽFig. 6.. Each group of cells will be described in turn. 3.1. RS, FS and IB cells The descriptions of RS, FS, and IB cells presented for other cortical areas Žsee Section 1. also apply to cells in auditory cortex. The descriptions here serve only to confirm the distinctive features of each cell type. RS cells were by far the most common cell type, comprising 160 of 187 Ž86%. classified cells. In response to 600 or 800 ms depolarizing current pulses of suprathreshold intensity, RS cells spiked for the duration of the pulse ŽFig. 1A, Ci.. The firing rate of RS cells adapted at the beginning of a pulse and then maintained a constant rate ŽRS1. or continued to adapt throughout the pulse ŽRS2; RS1,2 refer to subtypes of Agmon and Connors w2x.. While some RS cells adapted completely before the end of a near-threshold current pulse ŽFig. 1Ai., they generally spiked for at least 200–300 ms, and at higher intensities they always spiked for the duration of the entire pulse ŽFig. 1Aii.. Rates of adaptation for RS cells appear to form a continuum ŽFig. 1Ci., rather than delineating distinct RS1 and RS2 subclasses, thus, such distinctions

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were not made routinely. In response to large hyperpolarizing current pulses, RS cells often displayed a delayed depolarizing ‘sag’ suggestive of inward rectification Žnot shown; similar responses are evident in other cell types, see Fig. 3D, open arrowhead.. The passive membrane and spike characteristics for RS cells are in Table 1. Cells that were judged qualitatively to belong to RS1 and RS2 subgroups did not differ significantly in any of the properties listed in Table 1. FS cells were encountered in six cases ŽFig. 1B.. FS cells have narrow spikes ŽTable 1. that typically are terminated by large, fast-onset and short-duration afterhyperpolarizations ŽAHPs, see first spike in Fig. 1Bi.. FS cells can discharge at high frequencies and their firing does not adapt during a maintained stimulus ŽFig. 1B, Cii.. IB cells respond to depolarizing current pulses with a short burst of spikes, and sometimes burst repetitively and rhythmically throughout the pulse w2,39x. These cells can be found in layer IV w32x but more often are found in layer V w9,10,31x. We encountered only one IB cell in this population Žnot shown., located in layer IV. Twenty-five neurons filled with biocytin were recovered. Each of 11 RS cells were pyramidal neurons ŽFig. 1Aiii.. Only one FS cell was filled with biocytin, and this cell was nonpyramidal ŽFig. 1Biii.. The sole IB cell was recovered and found to be pyramidal. The other filled neurons either could not be classified physiologically Ž n s 6. or belong to one of the two cell types described next Ž n s 6.. 3.2. Rectifying (RECT) cells Two groups of neurons could not be placed into the previous categories. The first group, RECT cells, comprised 16 neurons with three related defining characteristics: Ži. RECT cells spiked early in response to current pulses and then stopped, typically within ; 250 ms ŽFig. 2A,B.. Žii. With increasing-amplitude depolarizing steps, the duration of the spike train either decreased ŽFig. 2Aii. or remained relatively constant ŽFig. 2Bii., even as the firing frequency increased. Žiii. Increasing-amplitude depolarizing steps produced decreasing increments of membrane depolarization Žsee traces in Fig. 2Ai, Bi, current step increments are of equal magnitude; see also I–V plots in Fig. 2Bv and C.. Membrane depolarization in RECT cells could activate an outward current to suppress spiking behavior and reduce Vm deflections. Evidence for such a phenomenon can be seen in subthreshold voltage depolarizations. For the cell in Fig. 2B, depolarizing current steps of sufficient amplitude produced an initial rapid depolarization that ‘sagged’ within tens of ms to a more negative potential ŽFig. 2Biv, open arrowhead.. Such a sag could result from activation of an outward Žhyperpolarizing. current, or from inactivation of an inward Ždepolarizing. current. However, the suppression of longer-latency action potentials with

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Fig. 1. Characteristics of pyramidal RS cell and nonpyramidal FS cell in rat auditory cortex. A. RS intrinsic physiology in a neuron from a P11 animal. Current pulses Žnot shown. were y0.2 to q0.1 nA in steps of 0.1 nA in Ži. and q0.2 nA in Žii.. Resting Vm y68 mV. Žiii. Biocytin-filled layer IIrIII pyramidal neuron whose physiology is shown in Ži. and Žii.. B. FS intrinsic physiology in a P14 neuron. Current pulses were y0.1 to q0.3 nA in steps of 0.1 nA in Ži. Žresponse to subthreshold 0.2 nA stimulus not shown, for clarity of viewing spikes. and q0.4 nA in Žii.. Resting Vm y68 mV. Žiii. Layer IIrIII nonpyramidal neuron whose physiology is shown in Ži. and Žii.. C. Ži. Plots of spike number vs. time of occurrence indicate varying degrees of adaptation to just-suprathreshold current pulses in 13 RS neurons. Asterisk indicates plot based on response in Aii. Žii. Plots of spike number vs. time of occurrence are given for five stimulus intensities Ž0.3–0.7 nA. for interneuron in B. Asterisks indicate plots based on responses in Bi and Bii.

ever-greater depolarizing steps Že.g., Fig. 2Aii. strongly implies the presence of hyperpolarizing current. Also, the presence of an AHP following the end of the subthreshold current pulse ŽFig. 2Biv, closed arrowhead. whose ampli-

tude increased with increasing membrane depolarization could reflect the tail of an outward current. Thus, depolarization-induced outward rectification likely contributes to the characteristic physiology of RECT cells.

Table 1 Passive and spike characteristics of cells Žmean" S.E.M..

RS, n s 160 c FS, n s 6 RECT, n s 16 OS, n s 4 a

Vm ŽmV. a

R i ŽM V .

t Žms.

Spike threshold ŽmV. a

Spike height ŽmV. b

Spike width Žms.

y67 " 0.5 y60 " 3.3 y68 " 1.3 y62 " 1.4

216 " 7.1 197 " 37.1 223 " 9.6 202 " 66.7

19.7 " 0.4 19.0 " 4.4 23.8 " 1.3 20.4 " 4.6

y40 " 0.3 y37 " 2.2 y38 " 1.5 y45 " 2.2

71 " 0.6 53 " 4.0 64 " 1.8 59 " 1.1

1.1 " 0.02 0.6 " 0.03 1.1 " 0.08 1.0 " 0.17

Values not corrected for junction potential of ; y13 mV, see Section 2. Spike heights, especially for FS cells, are underestimated due to 5 kHz sampling rate. c Actual number of RS cells for each mean ranged from 155 to 160. b

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Fig. 2. Characteristics of RECT cells in auditory cortex. A. Ži. Superimposed voltage responses of a P14 neuron to current steps of "0.2 nA in increments of 0.05 nA; Vm y66 mV. Suprathreshold responses in Ži. are depicted separately in Žii.; note that the decrease in spike amplitude during each response occurred only infrequently in RECT cells Žcf. response in B.. Žiii. Layer III nonpyramidal neuron whose responses are shown in Ži. and Žii.. B. Ži. Responses of a P12 neuron to current steps of y0.2–0 nA in increments of 0.05 nA, and 0.04–0.32 nA in steps of 0.04 nA; Vm y70 mV. Žii. Plot of spike number vs. time of occurrence for each suprathreshold response in Ži.. Žiii. Layer IV pyramidal neuron whose responses are in Ži.. Živ. Subthreshold responses Žcurrent steps of 0.05 and 0.1 nA. in same cell demonstrate hyperpolarizing sag Žopen arrowhead. upon sufficient membrane depolarization, and afterhyperpolarization Žclosed arrowhead. following the stimulus. Žv. I–V function based on records in Ži.; measurements made at end of current pulses. C. Mean I–V function for the 16 RECT neurons.

The strong spike frequency adaptation in RECT cells might also involve a Ca2q-dependent AHP activated by spike-induced depolarization w28,59x. In general, increasing-amplitude depolarizing steps produced increased number of spikes Žalthough the suppression of longer-latency action potentials often meant that the increased number of spikes occurred within a shorter duration train. and increased amplitude AHPs Žnote, however, that not all RECT cells had obvious AHPs; see Fig. 2Ai.. Thus, the AHP magnitude increased both with the number of spikes and with depolarization. However, in a few cases the effects of spike number and depolarization could be dissociated. As shown in Fig. 2Bii, increased depolarization did not always elicit an increased number of spikes Žcurrent steps of 0.2 and 0.24 nA each elicited four spikes, and steps of 0.28 and 0.32 nA each elicited five spikes.. In each case that the number of spikes remained constant with increased

depolarization, the larger depolarization produced an approximately 0.5 mV larger amplitude AHP Žnot shown.. Thus, spike-induced depolarization likely contributes to, but does not entirely account for, AHP magnitude, consistent with the data obtained from subthreshold depolarizing responses ŽFig. 2Biv. suggesting that a separate outward current is active in RECT cells. Fig. 2C depicts the mean I–V response for the 16 RECT cells, obtained by measuring voltage responses near the end of each current pulse. The dashed line is extrapolated from the data points near the resting potential, and emphasizes the nonlinear portion of the I–V function that begins near y55 mV. Note also the lesser rectification at hyperpolarized potentials Ž- y80 mV. that is also visible in the voltage traces in Fig. 2A,B. Such rectification often was associated with a depolarizing sag following an initial large hyperpolarization Že.g., Fig. 2Bi..

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Four of the 16 RECT cells were filled with biocytin and recovered. Of these, two were nonpyramidal neurons ŽFig. 2Aiii. and two were pyramidal ŽFig. 2Biii.. 3.3. On-spiking (OS) cells The second group of nonconventional cells resembled RECT cells in that they showed evidence for outward rectification upon membrane depolarization. However, they differed sufficiently in two respects to be considered separate from RECT cells: Ži. OS cells fired only one or two spikes in response to depolarizing pulses, and ii. the latency to the spike onset Žthreshold. was unusually short, i.e., - 10 ms. We encountered four of these cells and refer to them as on-spiking cells.

Data from three of the four OS cells are shown in Fig. 3 and illustrate their defining characteristics. In each case, depolarizing current pulses of threshold intensity elicited a single spike within 10 ms Žmean 7.8 " 1.0 ms, n s 4.. With increased-amplitude depolarization, spike latency decreased slightly, and in one case, a second spike appeared ŽFig. 3Dii; a third level of depolarization in this cell again elicited two spikes at even shorter latency.. Overlapping traces in Fig. 3Ai, Ci and Di demonstrate the temporal consistency of evoked spikes Žthere is one spike at each of two levels in Fig. 3Ai, and 1–2 spikes at each of three levels in Di. and evidence for membrane rectification in both depolarizing and hyperpolarizing directions Žcurrent steps increments are of equal magnitude.. Traces for each cell are shown at higher resolution in Fig. 3Aii, Cii, and

Fig. 3. OS cells in layer IV of auditory cortex. A. Ži. Superimposed voltage responses of a P13 neuron to current steps of y0.2 to q0.4 nA in increments of 0.1 nA; Vm y60 mV. Note that the cell fired a single spike in response to each of the two largest current steps. Responses to depolarizing current steps in Ži. are shown at higher resolution in Žii.; traces with truncated spikes are offset for clarity. B. Mean I–V function for the four OS neurons. C. Ži. Responses of a P11 neuron to current steps of "0.2 nA in increments of 0.05 nA; Vm y66 mV. Responses to steps of y0.05 to q0.2 nA in Ži. are shown at higher resolution in Žii.; note the rapid activation of outward rectification upon subthreshold depolarizing steps. Although this cell spiked at only one level, it was included in the OS group because of its single spike, short spike latency and outward rectification. Žiii. Layer IV pyramidal neuron whose responses are in Ži. and Žii.. D. Ži. Responses of a P12 neuron to "0.2 nA in increments of 0.05 nA; Vm y60 mV. Open arrowhead indicates depolarizing sag in response to large hyperpolarization. Higher resolution responses in Žii. are in response to steps of 0.00, 0.02, 0.04, 0.06, 0.1, 0.15 nA. Closed arrowhead indicates robust spike AHP. Žiii. Layer IV nonpyramidal neuron whose responses are in Ži. and Žii..

R. Metherate, V.B. Aramakisr DeÕelopmental Brain Research 115 (1999) 131–144

Dii. Note the short latency to spikes, the large, fast spike AHPs Žarrowhead in Fig. 3Dii; traces with truncated spikes are offset for clarity., and in subthreshold responses, the rapid activation of rectification similar to that observed in RECT cells Žcf. Fig. 2Biv.. Note the similarity of spiking and rectifying properties within the OS group of cells, as well as the different degrees of hyperpolarization-activated rectification and depolarizing sag Že.g., arrowhead in Fig. 3Di.. Fig. 3B depicts the average I–V function for OS cells, obtained by measuring voltage responses near the end of each current pulse. Note the similarity of this function to that for RECT cells ŽFig. 2C. in that the deviation from a linear function in the depolarizing direction begins around y55 mV. Two of the four OS neurons were filled with biocytin. Of these, one was pyramidal ŽFig. 3Ciii. and one was nonpyramidal ŽFig. 3Diii.. 3.4. Comparison of features among cell groups The passive and spike properties listed in Table 1 were compared to identify possible functional distinctions among the four cell groups ŽANOVA and Scheffe’s F-post hoc procedure.. Neither resting Vm nor R i differed among the cell types Ž ps ) 0.05.. Membrane time constants did not differ among groups, except for the RECT vs. RS compari-

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son Ž p - 0.05.. Thus, passive properties largely did not differ among the cell groups. Comparison of spike properties produced unremarkable distinctions except in one case. The mean FS spike width of 0.6 ms was significantly less than the ; 1 ms width of spikes from the other cell classes Žone-tailed t-test vs. RS and RECT, ps - 0.001 vs. OS, p - 0.05.. While spikes heights also differed among cell groups, note that the 5 kHz digitization rate used in this study produced truncated FS spikes, since these spikes are unusually fast. This resulted in underestimated spike heights and overestimated spike durations Žsince duration is measured at half amplitude.. Thus, while the narrower widths of FS cells are undoubtedly real Žand even narrower than reported., the lesser spike heights are at least partly artifactual. Measures for the other cell classes, with spike durations nearly twice as long, were less affected by digitization rate. Spike thresholds differed significantly Ž p - 0.05. between FS and OS cells and between OS and RECT cells, but in general appeared similar among the groups. The most striking distinction among spike features, therefore, is the shorter spike duration of FS cells. An important criterion used to distinguish among cell types was the duration of the spike train elicited by suprathreshold current pulses. To demonstrate the usefulness of this criterion, Fig. 4A displays examples for each cell class Žthe analyses in Fig. 4 utilized all 10 FS and OS

Fig. 4. Comparison of firing durations and latencies to first spike among cell groups. Analyses in A and B utilized all 10 FS and OS cells, as well as all RS and RECT cells Žnine cells each. with data from suprathreshold stimulus intensities at three or more current levels. Each line depicts the effect of changes in current intensity on firing duration ŽA. or latency to first spike ŽB. for a single cell.

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R. Metherate, V.B. Aramakisr DeÕelopmental Brain Research 115 (1999) 131–144

cells, as well as all RS and RECT cells Žnine cells each. with data from suprathreshold stimulus intensities at three or more current levels.. For each cell, lines connect points obtained at different current levels. While RS and FS cells sometimes did not spike throughout a current pulse at threshold, they always spiked throughout the pulse at higher intensities ŽFig. 4A.. In contrast, RECT cells tended to spike only for the first 100–300 ms at threshold, and the duration of spiking either decreased or stayed relatively constant with increases in intensity. The single OS cell that produced two spikes Žallowing measurement of a spike train duration. is shown for comparison. Thus, the duration of cell spiking effectively distinguished among some cell groups. One criterion that defined OS cells was the spike latency at threshold current intensity. The latency to first spike for OS cells Ž7.8 " 1.2 ms. was significantly less than that for RS cells Ž85 " 6.4 ms, one-tailed t-test, p - 0.05. and RECT cells Ž57 " 11.6 ms, p - 0.05., but not less than the latency for FS cells Ž116 " 52.7 ms, p ) 0.05., due to the large variability in the FS group. Data for each cell group showing the change in first spike latency with current intensity are in Fig. 4B Žfor clarity, only latenciesF 40 ms are shown; data are from same cell sample as in Fig. 4A.. The faster onset of OS spikes clearly distinguishes that group from the others. It is possible that the shorter spike latency of OS cells results from outward rectification suppressing first spikes that otherwise would appear at longer latencies. If so, then only stimuli that were sufficiently strong to elicit spikes before the onset of outward rectification would be effective. We would predict, therefore, that threshold current intensities for OS cells would be higher than for other cell groups. Consistent with this possibility, the threshold stimulus intensity for OS cells was 0.19 " 0.04 nA; this was significantly higher than the threshold for RS cells Ž0.05 " 0.01 nA, p - 0.001. and RECT cells Ž0.09 " 0.01 nA, p - 0.01., but not FS cells Ž0.12 " 0.04 nA, p ) 0.05; comparisons based on the 28 cells in Fig. 4.. RECT cells also displayed outward rectification which might be expected to raise their thresholds, and, in fact, threshold intensities for RECT cells were higher than for RS cells Ž p - 0.01.. Thus, outward rectification may suppress longer latency spikes, leading to shorter-latency threshold responses at higher stimulus intensities. While these data suggest that the short spike latencies of OS cells result from outward rectification, other factors may also contribute since at a fixed suprathreshold current level Ž0.2 nA., OS cells still spiked at shorter latencies than RS and RECT cells ŽOS 5.6 " 1.6 ms vs. RS 11.3 " 0.9 ms, p - 0.01 vs. RECT 15.7 " 2.5 ms, p - 0.05.. These data suggest mechanistic bases for the unusual characteristics of OS and RECT cells. OS and RECT cells appeared similar in terms of Ži. outward rectification, Žii. a lack of spikes at longer latencies, and Žiii. higher threshold stimulus intensities. We

therefore looked for mechanisms that might account for their differences. Specifically, we predicted that the lesser number of spikes Žone or two. elicited at shorter latencies Ž- 10 ms. in OS cells could result from faster activation of outward rectification. In 11 cells Žfour OS, seven RECT. where a clear hyperpolarizing sag implied the onset of outward rectification at a subthreshold membrane potential Že.g., Fig. 2Biv, open triangle., we measured the latency to the peak depolarization from which the sag began. The peak latency for OS cells was 21.8 " 5.8 ms, significantly faster than the latency of 46.6 " 6.9 ms for RECT cells Žone-tailed t-test, p - 0.02. and consistent with our prediction. There was no difference between the Vm of the peak depolarization ŽOS y48 " 2.0 mV, RECT y45 " 2.5 mV, p ) 0.05., indicating that the rate of depolarization was faster in OS cells than in RECT cells. Thus, faster outward rectification may distinguish OS from RECT cells and could contribute to functional differences between the two groups; however, voltage-clamp studies will be necessary to demonstrate this conclusively. Rectification in OS and RECT cells could also influence cell responsiveness by modifying passive membrane

Fig. 5. Membrane depolarization strongly reduces R i and t for OS and RECT cells Žcombined.. Values for R i and t are based on measurements made at the end of current pulses, to ensure full activation of voltage-dependent currents, except for measures attributed to resting Vm which were made at the beginning of a y0.1 nA pulse. Data points were grouped by current level Ž"0.2 nA in 0.05 nA steps. and values for Vm , R i and t were averaged for each level. Except for data at y101 mV, error bars for Vm are smaller than symbols used.

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properties. Fig. 5 illustrates how R i and t varied with Vm for the 20 OS and RECT cells Žgroup data combined due to similar degrees of rectification.. From resting Vm Žy67 mV. to approximately y45 mV, R i decreased 47% from 219 " 14.2 to 116 " 6.0 M V Žpaired t-test, p - 0.001., and t decreased 42% from 23.1 " 1.3 to 13.4 " 1.2 ms Ž p - 0.001.. Such changes could strongly influence neuronal responses by reducing the amplitude, time course, and spatial integration of synaptic inputs. 3.5. Correlation of cell types defined morphologically and physiologically Of the 25 cells filled with biocytin and recovered, 19 were unambiguously classified with respect to intrinsic physiology. As reported above, each of the 11 RS cells filled with biocytin was pyramidal. These findings are

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consistent with assessments of neuron morphology made upon visualization with IR-DIC optics during experiments. The vast majority of RS neurons had prominent apical dendrites that ascended towards the pia, i.e., these neurons clearly were pyramidal. The single biocytin-filled FS cell was nonpyramidal, consistent with the round somas and lack of apical dendrites of FS cells visualized with IR-DIC optics. OS and RECT cells filled with biocytin were found to be either pyramidal or nonpyramidal, indicating that these physiological response types are not associated exclusively with either broad morphological category. It is of interest, however, that in the case of two cells Žone OS, one RECT., a preliminary classification during the experiment of ‘potentially nonpyramidal’ Žbased upon soma shape and the lack of a prominent apical dendrite. was demonstrated incorrect by the biocytin results. It is possi-

Fig. 6. Changes in passive membrane and spike properties during development. Data from all neurons recorded in this study are included. Correlation coefficients indicate significant correlation of resting Vm , R i , t , spike height and spike width, but not spike threshold, with age over the range of P8–21. Data were grouped for P8–9 Ž n s 11 neurons., P10–11 Ž34., P12–13 Ž89., P14–15 Ž52., P16–17 Ž17., P19–21 Ž23; no data from P18. and mean values" S.E.M. were plotted at the first day for each age range.

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ble that finer morphological distinctions Že.g., less prominent apical dendrites. may distinguish OS and RECT pyramidal neurons from other pyramidal neurons. Larger samples will be required to resolve this issue. It would be useful to determine the laminar distribution of the cell types described here, however, a likely sampling bias precludes definitive answers. The majority of RS cells were found in layer IIrIII of the cortex, whereas nearly all OS and RECT cells were located in layer IV or near the border between layers III and IV. However, we made no attempt to sample different cell types in each layer equally, and post hoc analyses suggest that experiments were biased towards recording either from pyramidal neurons in layers IIrIII or from smaller, putative nonpyramidal neurons in layer IV. Thus, while OS and RECT cells appear to lie preferentially in layer IV, they may instead tend to be smaller neurons which were mostly sampled in layer IV. Nonetheless, one implication of these data, along with IR-DIC and biocytin results Žprevious paragraph., is that OS and RECT cells may include nonpyramidal neurons as well as small pyramidal neurons with slender apical dendrites. 3.6. Changes in neuron characteristics with deÕelopment Neurons examined in the present study were obtained from animals aged P8–23, a period of significant functional development Žsee Section 1.. Several physiological parameters were found to vary with age, as shown in Fig. 6. Resting Vm , R i and t all decreased with age. Spike amplitude increased with age and spike width decreased. Spike threshold did not change, nor did the latency to first spike at threshold stimulus intensity Žnot shown; p ) 0.05.. These changes in passive and spike properties may contribute to the functional maturation of auditory cortex. The relative prevalence of cell types may also change with age. In the interval between ages P11–15, we encountered 111 RS cells and all 20 OS and RECT cells. In contrast, during P16–20 there were 24 RS cells and no OS or RECT cells. The distribution of cell types across these intervals differs significantly Žchi-squared, p - 0.05; OS and RECT cells were combined because of their similar rectification properties and low numbers.. Interestingly, whereas outward rectification became less prevalent with development, some cells from older slices Že.g., P20. displayed clear inward rectification upon depolarization Žnot shown.. It is possible that OS andror RECT cells may function only during development, however, the low number of cells studied at greater ages precludes strong conclusions. Additional studies on adult tissue will be required to resolve this issue. 4. Discussion In this study we report on the intrinsic electrophysiology, including spiking patterns, of neurons in rat auditory

cortex during the second and third weeks after birth. Many neurons display intrinsic physiology and morphology consistent with previous descriptions of response classes ŽRS, FS, IB neurons.. In addition, we identify two groups, RECT and OS neurons, that are characterized by briefer spike trains in response to maintained depolarizations, and by strong depolarization-induced outward rectification. Unusually brief Ž1–2 spikes. and fast Ž- 10 ms spike latency. responses further distinguish OS cells from RECT cells. Biocytin labeling demonstrated that RECT and OS cells can be either pyramidal or nonpyramidal neurons. The intrinsic electrophysiology of different cell groups may contribute to auditory cortex functions. 4.1. Mechanisms of intrinsic electrophysiology The intrinsic electrophysiology of a neuron reflects the types and distributions of membrane ion channels. The collective behavior of these channels determines how a neuron responds to depolarizing and hyperpolarizing current pulses and, presumably, to synaptic inputs w10,38x Žbut see Ref. w45x.. Intrinsic electrophysiology is therefore likely to be an important determinant of neuronal responsiveness. 4.1.1. Regular spiking neurons The spike discharge of RS cells adapts to a maintained depolarizing current pulse. The adaptation is thought to involve spike-induced activation of a Ca2q-dependent Kq current w28,30,59x. Previous studies have subdivided RS cells depending on their rate of adaptation w2x. However, in the present and previous studies, rates of adaptation appear to form a continuum with the RS1 and RS2 subdivisions representing extremes. For some whole-cell recordings in the present study, rates of adaptation decreased over time Žtens of minutes., probably reflecting ‘washout’ of an intracellular constituent Že.g., Ca2q buffer or free Ca2q .. Similarly, varying rates of adaptation in different neurons may reflect varying intracellular levels of the same constituentŽs.. Some RS cells adapt completely, well before the end of a near-threshold current pulse, and in this respect they resemble RECT cells. However, with an increase in stimulus intensity RS cells always spiked for the duration of the current pulse. Further, they did not display membrane rectification with increasing depolarization. These characteristics distinguished RS and RECT cells, and indicate that different mechanisms underlie the two types of cell properties Žsee below.. 4.1.2. Fast spiking neurons The narrow spike width of FS cells that gives rise to their name results from the unusually rapid repolarization of the action potential. Previous studies have shown that the rate of rise of FS spikes is similar to those of other cell types, but the falling rate is significantly faster w32x. The spike’s fast AHP also permits FS cells to discharge at a

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high rate, up to several hundred Hz, in response to maintained depolarization. The lack of adaptation in FS cells may indicate the lack of a Ca2q-dependent AHP, or perhaps an intracellular Ca2q buffer Že.g., parvalbumin w22x. that prevents activation of a slow AHP. The relative lack of FS cells in the present sample is puzzling, for although their scarcity in previous studies has been attributed to their small size w10x, size is not a major factor in these experiments due to the IR-DIC optics. Other factors might explain their low numbers, such as an increased vulnerability to injury so that FS cells do not survive near the cut surface of the slice where most cells are recorded. Also, in the present study many recordings were targeted at pyramidal rather than nonpyramidal neurons Žsee Section 3.. Given their probable GABAergic nature w23,64x Žbut note that some nonadapting cells with fast spikes are pyramidal neurons w14,62x., the scarcity of FS cells in the present study is unlikely to reflect either their true numbers or their functional importance.

4.1.3. Rectifying neurons The major defining feature of RECT cells was striking outward rectification that began within ; 50 ms of a depolarizing stimulus pulse and appeared to strongly suppress spike discharge. Examination of the AHP after subthreshold responses indicated that the magnitude of the outward Žhyperpolarizing. current increased with increasing amplitude depolarizing steps. This voltage-dependence of the outward current may underlie the observation that the duration of spiking decreased with larger depolarizing steps, i.e., greater depolarization could produce a stronger outward current that would suppress firing more strongly. Since rectification and the AHP occurred in subthreshold responses, spike-induced mechanisms Že.g., Ca2q-dependent AHP. cannot wholly account for the observed effect. Many cells that were not classified physiologically because they could not be placed easily into a response class had properties that overlapped the RECT and RS Žspecifically, RS2. groups, indicating that the frequency of the rectifying current is likely underestimated. Further, it may be that the degree of rectification across neurons forms a continuum, rather than defining two distinct classes. The functions of outward rectification, however, should be most clearly discerned in RECT neurons. Outward Kq currents have been implicated in strong spike adaptation in previous studies of cortical neurons. In sensorimotor cortex slices from rats 1- to 4-weeks-old, many neurons spike only for the first 100–250 ms of a 1 s depolarizing step Ždesignated ‘completely adapting cells’; Ref. w28x.. The discharge is followed by a slow AHP that lasts for several hundred ms and produces a sag in the response to the depolarizing step. At times, the AHP ends before the end of the current pulse and additional spikes may recur. The addition of Ca2q channel blockers or neuromodulators blocked both the AHP and spike adapta-

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tion. In visual cortex neurons isolated from 2-week-old rats and maintained in culture, depolarizing steps revealed robust spike frequency adaptation w27x. In 24% of neurons, obtained from pups G P11, spike discharge adapted completely. The duration of spiking increased with increasing amplitude depolarizing steps, but after cessation of spiking the Vm held steady for the remaining duration of the current pulse, i.e., the response did not resemble a slow AHP. Pharmacological and voltage-clamp manipulations implicated Ca2q-independent Kq channels in the spike frequency adaptation. Thus, in other preparations of sensory cortical neurons, both Ca2q-dependent and Ca2q-independent Kq currents can produce spike frequency adaptation and rectification similar to that seen in RECT cells. Other studies of neurons from sensorimotor cortex of young Ž1–4 week. rats have emphasized inward, rather than outward, rectification upon depolarization from rest w25,34x. This inward rectification grows more prominent with age and is reduced by Naq channel blockade, implicating activation of a persistent Naq current. Interestingly, in those studies, Naq channel blockade reveals striking outward rectification. In the present study, outward rectification was only observed at ages F P15, and inward rectification was most apparent in older neurons Ž; P20.. Thus, channels mediating depolarization-activated persistent inward and outward currents may coexist in the same neuron and may be regulated differently during development.

4.1.4. On-spiking neurons The major defining feature of OS cells was their short latency Ž- 10 ms. and brief spike response Ž1–2 spikes. to depolarizing pulses. Similar features are found in some brainstem auditory neurons w47,65x, but to our knowledge have not been described previously for cortical neurons. OS cells also displayed depolarization-activated outward rectification that began within ; 20 ms of stimulus onset, significantly faster than the nearly 50 ms latency to activation in RECT cells. While other aspects of the rectification in OS and RECT cells appeared similar, it seems unlikely that OS cells are simply an extreme case of RECT cells. For example, individual OS cells demonstrate weaker rectification Žalbeit faster. than some RECT cells Žcf. Figs. 2 and 3., which is inconsistent with the notion of OS cells being extreme cases. Further, the duration of RECT cell firing Že.g., 100–250 ms. was always considerably longer than OS cells, i.e., there was no continuum of firing durations between OS and RECT cells. Finally, even at high stimulus intensities RECT cells generally did not spike at as short a latency as did OS cells at their threshold. Thus, other factors, possibly morphological as well as physiological, determine OS firing behavior. Some brainstem auditory neurons exhibit rectifying Kq currents that limit to 1–2 spikes the neuron’s responses to either depolarizing current pulses or afferent EPSPs

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w4,47,53,65x. One mechanism underlying this behavior is a low threshold Kq current that activates rapidly, within a few ms upon depolarization from rest, and thereby limits spike discharge w4,47x. Pharmacological reduction of this current results in multiple spikes being elicited at a lower current step intensity. It has been suggested that the relevant Kq channels may be clustered near the neuron’s spike initiation zone to more effectively regulate spike discharge w4x. Similar Kq currents in cortical neurons may contribute to the fast, brief spiking in OS cells and to rectification in both OS and RECT cells. OS cells comprised a very small group within the present study, however, it is unclear whether this reflects a sampling bias or is a reflection of their true numbers. OS cells may be preferentially distributed, e.g., among smaller neurons in layer IV, and therefore difficult to sample equally with larger pyramidal neurons. Other factors may also contribute to the scarcity of OS cells. As described above, recordings from FS cells are also scarce, yet these putative GABAergic neurons have an undisputed functional importance. Similarly, the importance of OS cells may be disproportionate to their numbers in the current study. Finally, it should be noted that OS and RECT cells are not an artifact of the particular recording conditions used here, since they are encountered in slices maintained in an interface-type recording chamber and in recordings using either patch pipettes or sharp microelectrodes ŽS. Cruikshank and R. Metherate, unpublished data.. 4.2. Intrinsic currents actiÕated by membrane hyperpolarization Numerous cells exhibited nonlinear behavior upon membrane hyperpolarization. Within tens of milliseconds following large hyperpolarizing steps, in particular, a depolarizing sag occurred frequently in RS, RECT, and OS cells Že.g., Fig. 2B, Fig. 3D.. This behavior has been studied extensively in thalamic and cortical neurons and represents activation of the mixed cation current, I h w33,60x. This current likely contributes to spontaneous slow oscillations in the intact animal as well as to recovery from long-lasting Kq-dependent IPSPs. Intrinsic currents activated by membrane hyperpolarization were not a focus of this study, and more extensive discussions of their functions are found elsewhere w61x. 4.3. Functions of intrinsic electrophysiology in auditory cortex The most straightforward prediction of the functional relevance of intrinsic properties described here is that different cell classes will respond differently to sustained depolarizing inputs. Thus, RS pyramidal neurons would fire moderately upon depolarization and excite nearby and distant neurons via glutamatergic axon collaterals w35,64x.

Inhibitory FS cells would fire strongly upon excitation and thereby produce a barrage of IPSPs on nearby cells w5,24,64x. RECT cells could fire strongly, but only for a limited time, after which their output would be suppressed, and further depolarization opposed by membrane rectification. The intrinsic physiology of OS cells suggest several functional implications. In brainstem auditory nuclei, neurons with similar electrophysiology are able to precisely encode the timing of afferent inputs w47,65x. It is intriguing to consider similar roles for cortical OS cells. For example, OS cells may signal the timing of stimulus onsets. Recent studies of cortical function stress the importance of detecting stimulus transients w16,50x, and OS cells are ideally suited to mediate such a function. Appropriate synaptic contacts with other cells could contribute to the well known tendency for cortical neurons in vivo to fire transiently to maintained stimuli Žsee Introduction.. Similarly, if OS cells receive afferent inputs from diverse sources, their single spike discharge could signal synchronous or coincident inputs, and even enhance synchrony by suppressing nonsynchronous inputs. For example, following spike discharge, outward rectification in OS Žand RECT. cells would reduce the impact of subsequent EPSPs as a result of the greater than 40% decrease in R i and t . The faster time course of afferent EPSPs might also be useful for timing the onsets of repetitive inputs. 4.4. Changes in intrinsic electrophysiology during deÕelopment Several intrinsic and spike properties Že.g., Vm , R i , t , spike height and width. changed over the developmental period studied ŽP8–23., as seen in previous studies of cortical neurons w25,28,34x. Electrophysiological properties change little between P20 and P30 w34x, suggesting that physiological maturation is largely complete by the end of the period covered in the present study. Firing patterns of cortical neurons are also regulated developmentally w25,28x. However, the impact of such changes, and the response patterns per se, on cortical development and function are unclear. Given that sensory experience and patterns of cortical activity are thought to guide the formation of functional synaptic circuitry during development w20x, OS and RECT cells may promote the influence of auditory transients in the shaping of cortical circuitry. The implications of their actions should be determined in future studies.

Acknowledgements We thank Dr. S. Cruikshank for helpful discussions and comments on the manuscript, and Ms. N. Patel for histological processing. This work was supported by the NSF ŽIBN 9510904. and the NIH ŽNIDCD, DC02967..

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