INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The auditory cortex (ACx) integrates and processes information carried along thalamocortical pathways to perform its major functions (for reviews, see de Ribaupierre 1997; Ehret 1997; Phillips 1995). Much is known about auditory thalamocortical pathways from anatomical and physiological studies. The main input to ACx is the primary thalamocortical pathway (often referred to as the lemniscal pathway) that projects from the ventral division of the medial geniculate (MGv) to the middle layers of primary ACx (Caviness and Frost 1980; Romanski and LeDoux 1993; Willard and Ryugo 1983; reviewed in Winer 1992). Physiological recordings from MGv and ACx indicate that this pathway mediates short-latency responses to acoustic stimuli and carries precise information about stimulus frequency, intensity, and timing (thalamus: Bordi and LeDoux 1994; Calford 1983; Edeline et al. 1999; Lennartz and Weinberger 1992; ACx reviewed in: de Ribaupierre 1997; Eggermont 1998; Ehret 1997). There are also nonprimary thalamocortical pathways (referred to as nonlemniscal or adjunct), including projections from the dorsal and medial divisions of the MG (MGd, MGm), the peripeduncular nucleus, and other nonprimary thalamic nuclei (Arnault and Roger 1990; Clerici and Coleman 1990; Herkenham 1980; LeDoux et al. 1985; Linke and Schwegler 2000; Ryugo and Killackey 1974). Typically these projections target superficial layers of temporal cortex. Responses of nonprimary thalamic cells tend to be weaker, have longer latencies, and more variability than MGv (Allon et al. 1981; Bordi and LeDoux 1994; Calford 1983; Edeline et al. 1999; Lennartz and Weinberger 1992), so their influence on cortex is expected to be more subtle (discussed in McGee et al. 1992; Sukov and Barth 2001; Weinberger 1995). Thus ACx function depends on the integration of both primary and nonprimary inputs.

Despite a large number of anatomical and physiological studies, very little is known about the cellular and synaptic mechanisms by which thalamic inputs are transmitted to, and processed in, ACx. Some progress has been made using in vivo intracellular recordings (e.g., Metherate and Ashe 1993; Mitani and Shimokouchi 1985; Sukov and Barth 2001), but such studies are hampered by the difficulty of making precise electrophysiological and pharmacological measurements in vivo. Yet detailed intracellular information must be obtained to address even seemingly simple questions such as how synaptic integration produces neuronal receptive fields. Brain-slice preparations offer an ideal environment to make precise intracellular and pharmacological manipulations, and considerable information has been obtained from ACx slices (Buonomano and Merzenich 1998; Hefti and Smith 2000; Kudoh and Shibuki 1997; Metherate and Ashe 1994). Nonetheless, the issue of thalamocortical processing has not been addressed directly because thalamocortical axons are severed during the preparation of conventional (coronal) slices and thus cannot be stimulated selectively.

To address similar problems in the somatosensory system, Agmon and Connors (1991) developed a slice preparation that maintained connections from the ventrobasal complex to barrel cortex. Since its development, that preparation has been applied to numerous issues fundamental to the understanding of thalamocortical processing in the somatosensory system. These include determining which cortical cells, as defined by position, morphology, and intrinsic properties, receive direct thalamic input (Agmon and Connors 1992; Gibson et al. 1999; Porter et al. 2001); what transmitters mediate thalamocortical transmission (Gil and Amitai 1996a); functional differences between intracortical and thalamocortical synapses (Gil and Amitai 1996b; Gil et al. 1997, 1999); and mechanisms of synaptic plasticity (reviewed in Castro-Alamancos and Connors 1997; Feldman et al. 1999).

To facilitate similar progress in the understanding of auditory forebrain mechanisms, we have developed a brain-slice preparation that maintains an intact auditory thalamocortical pathway. In initial studies using slices from rat and mouse, we demonstrated the likely feasibility of such a preparation by showing that stimulation within the thalamus or thalamocortical pathway could elicit cortical responses (Metherate and Cruikshank 1999). More recently, we have focused on the mouse brain because its smaller size permits more intact connections within a slice and for future transgenic studies. We have substantially refined the preparation to the point that we can reliably identify and study the primary auditory thalamocortical pathway in vitro. The present study represents an extensive anatomical and physiological characterization of this more refined preparation. We also introduce a second slice preparation, obtained from the area immediately ventral to the primary preparation, that may contain a nonprimary pathway. Several features of this second preparation are characterized and contrasted with those of the primary preparation. Portions of this study have appeared in abstract form (Cruikshank et al. 1999).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Preparation of slices

All procedures followed the University of California, Irvine, animal-use regulations. Slices were taken from postnatal day (P)13-P19 FVB mice and maintained in vitro. Slice planes were nearly horizontal, but with the lateral end typically raised 15°, as illustrated in Fig. 1A. This plane was chosen so that thalamocortical axons, and parts of the MGv and ACx, could all be obtained within a single slice (Fig. 1).

View larger version (103K): [in this window] [in a new window]   Fig. 1. Anatomical positions of the primary and shell slices. A: drawing on right shows schematic of coronal mouse brain section at level of medial geniculate (MG) and auditory cortex (adapted from Franklin and Paxinos 1997). The ventral MG (MGv, black) is surrounded by a shell of nonprimary auditory nuclei (gray). Likewise, primary auditory cortex (ACx, blackalso indicated by asterisk) is bordered dorsally and ventrally by nonprimary auditory cortical areas (gray). The dotted line shows the approximate 15° angle that allows both MGv and ACx to be obtained within a slice. A, left: the left hemisphere of an actual mouse brain, viewed from the front, illustrating the planes of the primary and shell slices; they are 600 µM thick, and cut at about the angle indicated in the adjacent drawing. Orientation: lateral toward right, dorsal toward top. B: same hemisphere as in A but viewed from lateral side. Orientation: anterior toward left, dorsal toward top. C: same as B except the cortex was dissected away, revealing midbrain and thalamic structures (SC, superior colliculus; IC, inferior colliculus). MG is visible, and its bottom 30-50% appears to be within the primary slice. Shell slice is below MG. The structure immediately anterior to MG is the lateral geniculate (LG). D and E: the primary and shell slices are laid down flat and viewed from the dorsal side (as in recording chamber). Orientation: anterior is toward left, lateral is toward bottom. Typical recording sites in the middle layers of temporal cortex are indicated by asterisks. The MGv (in primary slice) and a typical position for shell stimulation (in shell slice) are indicated by arrows. Dashed rectangles indicate approximate areas of the fluorescent images in Figs. 8, A1 and B1 (primary), and 10, A1 and B1 (shell).

Following decapitation under halothane anesthesia, brains were rapidly removed and placed in 0-4°C artificial cerebrospinal fluid (ACSF; in mM: 125 NaCl, 2.5 KCl, 1.25 KH₂PO₄, 25 NaHCO₃, 1.2 MgSO₄, 2 CaCl₂, and 10 dextrose; bubbled with 95% O₂-5% CO₂). Before slicing, the brains were blocked by making three cuts with a handheld razor blade while submerged in cold ACSF. First the anterior 25% of the brain was removed with a coronal cut. The remaining brain block was then propped forward to rest on its cut surface, and a second cut was made in a nearly horizontal plane (but 15° medial/lateral slant), which split the brain into dorsal and ventral portions. The dorsal portion was discarded, then the ventral portion was lifted from the ACSF and glued to the stage of a Vibroslice with its freshly cut surface facing down (i.e., dorsal surface down; Vibroslice from Campden, WPI; glue was Superglue, WPI). The glued brain block was immediately re-immersed in cold ACSF, and a third blocking cut was made, this time midsagitally, thus separating the left and right hemispheres.

The 15° slant on the second cut caused a greater portion of one of the hemispheres to remain intact (usually the left); it was this more intact hemisphere that was set to face the Vibroslice blade and from which slices were taken for recording. However, the "unused" hemisphere was also kept glued in place for support during slicing. Slices were cut in 400-600 µm sections starting near the ventral surface of the brain. As the region below the MG and ACx became visible, slices were retained, and placed either in the recording chamber or a holding chamber filled with ACSF at room temperature. Generally two to four slices were examined under a dissecting microscope (in recording chamber) to determine if they were in appropriate planes. If correctly blocked and sectioned, there would ultimately be two useful slices per brain (the "primary slice" and the "shell slice:" Fig. 1), each of which had distinct physiology and anatomy as presented in RESULTS.

In initial experiments (n = 29 slices), slices were cut at 600 µM, to ensure a high probability of intact thalamocortical connections. However, in later experiments, after learning more about the pathway trajectory, slice thickness was successfully reduced to 500 µM (n = 38; also n = 2 @ 400 µM), thus improving chances of maintaining healthy preparations.

Electrophysiology

Recordings took place in an interface chamber (Haas model, Med Systems) maintained at 34°C, with a liberal flow of warmed humidified gas (95% O₂-5% CO₂) and ACSF passing over the slices. An initial incubation period of 1-2 h preceded data acquisition.

Sharp intracellular recording microelectrodes were pulled from filamented glass (1.0 mm OD; A-M Systems) on a horizontal puller (p97, Sutter Instruments) and had resistances of 60-140 M when filled with 3 M K-acetate (+10 mM HEPES buffer, pH 7.3). Extracellular recording microelectrodes were also pulled from filamented glass (1.5 mm OD; A-M Systems), on the same puller, and had resistances of 0.5-5.0 M when filled with ACSF. Neural signals were amplified (CyberAmp AT-401 and Axoclamp 2B, Axon Instruments), monitored on oscilloscope (Tektronix), then digitized (5-20 kHz) and stored on computer (PowerMac, Apple Computer). Extracellular electrical stimuli (0.1-0.2 ms, 1-325 µA) were delivered via concentric bipolar electrodes (Ultrasmall, 25 µM inner core, 200 µM overall diameter, F. Haer) and a constant current isolation unit (Axon Instruments). Control of experiments and off-line analysis were done with computers (AxoData and AxoGraph, Axon Instruments; PowerMac, Apple Computer).

Stimulation and recording sites were visualized with a dissecting microscope (Fig. 1, D and E). Stimuli were delivered to the MG (primary slice, Fig. 1D) or the region ventral to the MG (shell slice, Fig. 1E, indicated by arrow). Responses were recorded in temporal cortex, from the cortical column with the largest extracellular response in layer 4 (i.e., the "focus"see Laminar response profile of primary slice is dominated by middle layer CSD sink). Intracellular recordings were mainly in layers 3-4 (30-50% of the distance from the pia to the white matter; asterisks in Fig. 1, D and E), and simultaneous extracellular recordings were usually in layers 3-4 for the primary slice, and layer 1 for the shell slice (<250 µM lateral distance between intra- and extracellular sites in layers 3-4). To determine the laminar locations of current sinks and sources, one-dimensional current source density (CSD) analysis was performed (Agmon and Connors 1991; Johnston and Wu 1995; Mitzdorf 1985). First, extracellular responses were recorded at evenly spaced locations (either 125 or 150 µM spacing) beginning two positions above the pia and ending either in the deep cortical layers or below layer 6. The CSD value for a given location (at a given time point) was then calculated by subtracting twice the voltage at that location from the sum of the voltages at the two nearest locations and dividing the result by the square of the distance separating recording sites.

To test for the roles of glutamate receptors in evoked responses, the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonopentanoic acid (APV; 50 µM) and the -amino-3-hydroxy-5-methylisoxazole-4-proprionic acid/kainate (AMPA/KA) receptor antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, 20 µM) were applied to the bath (both drugs from Research Biochemicals International). CNQX stock solution included dimethyl sulfoxide (DMSO; final concentration 0.4%). In experiments designed to block synaptic transmission, Ca²⁺ concentration in the ACSF was reduced and replaced with Mg²⁺ ("low calcium," in mM: 0.2 CaCl₂ and 3.0 MgSO₄, all other salts the same as normal ACSF). In experiments designed to selectively suppress polysynaptic responses (see INTRACELLULAR RECORDINGS IN LAYERS 3-4 OF THE PRIMARY SLICE REVEAL CONSISTENT EARLY RESPONSE AND MORE IRREGULAR LATE POLYSYNAPTIC RESPONSE), ACSF with high concentrations of divalent cations was used ("high divalent ACSF"; in mM: 115 NaCl, 2.5 KCl, 25 NaHCO₃, 4.2 MgCl₂, 7 CaCl₂, and 10 dextrose). Besides the adjustments in Ca²⁺ and Mg²⁺ concentrations (which were 3.5 times normal values), sulfates and phosphates were also left out to prevent Ca²⁺ precipitation (Crepel and Ben-Ari 1996; Luhmann and Prince 1990), and NaCl was reduced to maintain osmolarity (see preceding text).

Anatomy

FIXATION AND SECTIONING OF RECORDED SLICES. To examine the exact planes of section, some slices were processed for either Nissl staining or parvalbumin (PV) immunohistochemistry after electrophysiological recording (described in RESULTS). For both types of labeling, whole slices were first fixed by immersion in 4% paraformaldehyde (in 0.1 M Na-phosphate buffer: PB, pH 7.2) for 12 h, then sectioned on a vibratome at 50 µM.

PV IMMUNOHISTOCHEMISTRY. PV procedures were carried out with free-floating sections at room temperature with agitation, except where specified. Sections were pretreated for 30 min in 0.5% H₂O₂, rinsed in phosphate-buffered saline (PBS; pH 7.15; 0.1 M PB, 0.5 M NaCl) containing 0.1% Tween 20, then incubated for 2 h in PBS containing 0.3% Triton X-100. Next, nonspecific sites were blocked using an Avidin/Biotin Blocking Kit (Vector Labs), followed by immersion in 10% horse normal serum for 2 h (HNS, Vector Labs). Sections were then incubated at 4°C for 12-48 h in a combined antibody solution (to reduce background staining) (Hierck et al. 1994) containing a 1:500 dilution of monoclonal PV antibodies (pa-235, Sigma), a 1:500 dilution of biotinylated anti-mouse IgG (Vector Labs), 0.1 M PB, 0.5 M NaCl, 0.3% Triton X-100, 2% HNS, and 0.1% mouse normal serum (MNS, Sigma). Standard peroxidase visualization followed. Briefly, sections were rinsed in PBS containing 0.1% Tween-20, incubated in Avidin/Biotin complex for 2 h (Vectastain Elite ABC-Peroxidase Kit, Vector Labs), rinsed again in PBS, then in 0.05 M Tris buffer (TB; pH 8.0). Next they were preincubated in nickel/DAB solution (0.3% nickel ammonium sulfate, 0.033% diaminobenzidine, TB), then reacted for 2-5 min by adding H₂O₂ (0.01%). Following rinses in TB and 0.1 M PB, sections were mounted on gelatin-coated slides, air dried, dehydrated with increasing concentrations of alcohol, cleared with Histo-Clear (National Diagnostics), and coverslipped using Permount (Fisher).

NISSL STAINING. In the Nissl material, after sectioning at 50 µM (see preceding text), tissue was rinsed in 0.05 M PB, mounted on gelatin-coated slides, then air dried for 12-36 h. Next, the slide-bound sections were immersed for 2-24 h in a solution of 50% chloroform and 50% alcohol, rehydrated in descending concentrations of alcohol in H₂O, then immersed in 0.06% cresyl violet stain (0.6 mg/ml cresyl violet acetate, 0.72 mg/ml Na-acetate, 4.9 µl/ml 1 N acetic acid, H₂Ousually 300 ml) for ~5 min, followed by 30 s of rinsing in H₂O. After that, the tissue was dehydrated, cleared, and coverslipped as described for the immunoreacted tissue (see preceding text), except that acetic acid was added to the 95% alcohol dehydration step to accelerate differentiation of the cresyl violet stain (0.5 ml glacial acetic per 200 ml alcohol solution).

FIXATION AND SECTIONING OF WHOLE BRAINS. To compare Nissl and PV patterns in the coronal plane, sections were cut from whole fixed brains. Under deep anesthesia [50-100 mg/kg pentobarbital sodium (Nembutal)], mice were transcardially perfused with 0.9% saline until most visible blood was flushed from the animal, then with 4% paraformaldehyde (see preceding text) for ~10 min. Solutions were 4-8°C and perfused at 10-15 ml/minute using a peristaltic pump (Fisher). The brains were postfixed overnight at 4°C (same fixative), embedded in 3% agarose (to increase stability during sectioning), then sectioned on a vibratome at 50 µM in the coronal plane. Every third section was Nissl stained (see preceding text), and adjacent sections were immunoreacted for PV (see preceding text) and calbindin (not shown).

TRACT TRACING WITH Di-I. The fluorescent lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Di-I; D-282, Molecular Probes) was used to examine fiber pathways in previously recorded primary and shell slices and in some whole brains. First the slices and brains were fixed by immersion and perfusion respectively (described in the preceding text), then allowed to postfix 2 days. Next, 25- to 100-µM-diam particles of Di-I were inserted into targets in the still intact slices and brains under a dissecting microscope, using a broken micropipette to manipulate the Di-I (targets indicated in RESULTS). The slices or brains were then placed in small plastic cups and covered in warm liquid agarose (3%) that was made with PB containing 0.1% sodium azide. After the agarose hardened, the cups were filled to the top with PB (including 0.1% sodium azide), sealed with parafilm, and placed in the dark at 30-38°C for 1-4 mo to permit Di-I diffusion. The slices or brains were subsequently sectioned on a vibratome at 50 µM (for whole brains, sectioning was in horizontal plane), rinsed in PB, then mounted on gelatin-coated slides, and coverslipped with Vectashield mounting medium (Vector Laboratories).

SLICE PLANE EXAMINATION IN UNSTAINED WHOLE BRAINS. To compare the slice planes to the positions of the MG and other macroscopic features, six brains were fixed by perfusion (see preceding text) and cut on a vibratome in the planes normally used for physiological experiments. For clarity, no blocking was done so that the slice planes could be seen in the context of the whole brains. For the same reason, only three cuts were made on the vibratome: one cut separated the dorsal part of the brain from the primary slice, a second separated the primary and shell slices, and a third separated the shell slice and the ventral part of the brain (Fig. 1). The slice thicknesses were 600 µM and were in the approximate planes of physiologically recorded primary and shell slices, based on positions and appearances of structures contained within them (Fig. 1, D and E).

MICROSCOPY AND IMAGE ACQUISITION. The Nissl and PV labeling patterns were examined using standard bright-field microscopy at a variety of magnifications (×25-1,000), and images were captured with a digital camera (SPOT, Diagnostic Instruments) attached to an Olympus microscope. Di-I labeling was examined using epifluorescence microscopes, again at a variety of magnifications, and images were captured with digital cameras (Zeiss and Olympus). Photographs of the unstained tissue (Fig. 1) were taken using a Polaroid camera (MicroCam) fitted to a dissecting microscope (WPI), then the photo prints were digitized with a scanner (Color OneScanner, Apple Computer).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

As indicated in METHODS, when the mouse brain was cut along the near horizontal plane illustrated in Fig. 1, A and B, two distinct slice preparations could be obtained, each with unique physiological properties. The dorsal-most of these will be referred to as the "primary slice," and the more ventral will be called the "shell slice" (Fig. 1). Initially, the two preparations were distinguished based on gross anatomical features and by relating these features to the effective stimulation loci and cortical responses profiles. For example, in the unstained primary slice, the MG can be seen directly, and the lateral geniculate (LG), hippocampus, striatum, and other structures have characteristic shapes (Fig. 1D). When the MG is stimulated in a living primary slice, it results in a strong middle layer response in auditory cortex (described in the following text). The shell slice, and the structures contained within it, also have characteristic shapes. For example, the hippocampus is wider in the medial-lateral direction, giving it a more rounded appearance (Fig. 1E). More importantly, the shell slice is located in a sufficiently ventral plane that it contains no obvious MG nucleus. Thus in the shell slice, the region just medial to the hippocampus (indicated by an arrow in Fig. 1E) corresponds to the area ventral to the MG nucleus. Stimulation of this region consistently results in an upper layer current sink in temporal cortex (described in the following text), contrasting with the middle layer sink produced by MG stimulation in the primary slice.

Primary slice

ANATOMICAL FEATURES: PRIMARY SLICE CONTAINS MGV AND ACX. Figure 1C provides a lateral view of the mouse brain with part of the neocortex and hippocampus dissected away to facilitate comparison between the positions of the slices and the MG as a whole. The MG appears as a rounded protuberance posterior to the LG and ventral to the superior colliculus (SC). Notice that the primary slice contains the bottom 30-50% of the visible portion of the MG, which mostly consists of the core ventral division of the MG (MGv). Given that the slicing angle was chosen to obtain both the MGv and ACx within the same plane (Fig. 1A, right; see METHODS), it would seem logical that primary slices that contain the MGv would also contain primary ACx. These observed and inferred features of the primary slice, combined with its distinct middle layer response profile, are consistent with the known projection of the MGv to middle layers of primary auditory cortex (Caviness and Frost 1980; Romanski and LeDoux 1993; Willard and Ryugo 1983; reviewed in Winer 1992).

View larger version (90K): [in this window] [in a new window]   Fig. 2. Parvalbumin (PV) immunohistochemistry facilitates localization of auditory forebrain structures in thalamocortical slices. A: Nissl-stained coronal section (50 µM thick). Many structures within and surrounding the auditory thalamus can be differentiated in coronal plane using cytoarchitecture. The label "auditory cortex" is centered on approximate position of primary ACx. It is bordered dorsally and ventrally by nonprimary ("belt") areas (as indicated in Fig. 1A) (Franklin and Paxinos 1997). Besides position, primary ACx was identified by its more laminated cytoarchitecture (dense cell packing in layers 2-4 and 6, sparse in layer 5) than adjacent nonprimary areas, and the PV pattern (following text) (Cruikshank et al. 2001). Orientation: lateral toward left, dorsal toward top. B: PV pattern in section adjacent to A. Notice the dense PV labeling in MGv and sharp boundary along its ventral border (e.g., PP is virtually white). There is also dense and laminated PV labeling at the apparent position of primary ACx. The adjacent cortical areas have weaker PV labeling (e.g., region ventral to primary ACx but dorsal to rf has only lightly labeled somata). More ventrally, the perirhinal cortex (surrounding rf) has virtually no PV labeling. White lines in B (labeled 1-3) indicate the approximate planes of sections in C, 1-3. C: a primary thalamocortical slice was sectioned into 50-µM segments and PV immunolabeled. Orientation: anterior is toward left, lateral is toward bottom. The section in 1 was 100 µM from dorsal surface of slice, section 3 was the most ventral section of slice, section 2 was 50 µM above section 3. PV-immunopositive MGv is present in sections 1 and 2 but not 3. ACx expressed dense and laminated PV labeling in all 3 sections. Further described in RESULTS. APT, anterior pretectal nucleus; cp, cerebral peduncle; LG, lateral geniculate; MGd, dorsal division of MG; MGm, medial division of MG; PIN, posterior intralaminar nucleus; PP peripeduncular nucleus; rf, rhinal fissure; RT, reticular thalamic nucleus; SN, substantia nigra; str, superior thalamic radiation; VB, ventrobasal complex.

LAMINAR RESPONSE PROFILE OF PRIMARY SLICE IS DOMINATED BY MIDDLE LAYER CSD SINK. The dominant feature of the cortical response to MG stimulation in the primary slice is a robust negative field potential and associated CSD sink in layers 3-4. The example in Fig. 3A illustrates this and other typical features of the laminar response. In an initial procedure, the cortex was "mapped," by moving the electrode horizontally within layer 4, until finding the anterior-posterior position with the largest fast negative field potential evoked by MG stimulation (i.e., the "focus" of the response). The laminar profile was then determined for the cortical column corresponding to that focus by recording extracellular responses in evenly spaced intervals. Figure 3A1 shows the field potentials and CSD traces resulting from that procedure, conducted in normal ACSF. Following a 100-µA MG stimulus, the largest negative field potential and CSD sink (upward deflection) were recorded at 450 µM from the pia surface (total cortical thickness was 1,275 µM; Fig. 3A). Besides the large middle layer sink, the slice also displayed clear supragranular and infragranular CSD sources (downward deflections) at 150-300 and 750 µM, respectively. In addition, there was a small sink on the cortical surface.

View larger version (56K): [in this window] [in a new window]   Fig. 3. Laminar profiles and low-Ca2+ sensitivity of thalamocortical responses in the primary slice. A1: laminar field potentials and associated current source densities (CSDs) for example slice in normal artificial cerebrospinal fluid (ACSF). One-hundred-fifty-micrometer separation between recordings; numbers on left indicate depth of recording sites within ACx (gray background). "Pia" indicates surface of layer 1, "WM" indicates border between white matter and layer 6. MG stimulus intensity = 100 µA. Traces represent averages of 8-10 responses (gray-scale plot described in the following text). Notice large fast field potentials and CSD sinks around layers 3-4. Associated sources are immediately above and below. Also note small sink on surface; this was the smallest of the CSD responses classified as "surface sinks" (n = 6; see main text). A2: responses of same slice during perfusion of low-Ca2+ ACSF. Note the near absence of any evoked field potentials or CSDs. Responses recovered after return to normal ACSF (not shown). B: gray-scale CSD profiles of all primary slices, arranged by recording date. Areas under the raw traces were first measured (initial 20 ms after artifact), then gray-scale plots were generated using DeltaGraph (DeltaPoint). Values were normalized to the maximum sink for a given slice (max sink = +1), and plotted from +1 (black) to 1 (white), with 20 linear gray steps between (scale in B). Plot heights normalized to total cortical thickness. Some slices were not recorded to the white matter, so they don't reach 100%. Note generally consistent sink positions (dark bands) in layers 3-4. For further explanation, see RESULTS.

PRIMARY SLICE RESPONSES REQUIRE SYNAPTIC TRANSMISSION: CALCIUM SENSITIVITY. Blocking synaptic transmission, by lowering Ca²⁺ concentrations in the ACSF (replaced with Mg²⁺; see METHODS), profoundly suppressed the MG-evoked responses across all cortical laminae. Figure 3A2 illustrates this effect. After confirming the typical middle layer profile in normal ACSF (Fig. 3A1), low calcium ACSF was perfused over the slice for ~1 h (until reductions in layer 4 field potentials had become asymptotic), then the laminar profile was re-determined. Figure 3A2 shows that the MG-evoked field potential, and CSD traces are almost flat under low calcium conditions, with only extremely weak responses in the lower layers remaining (600-1,200 µM deep). Four other primary slices were tested in this way. All had a virtually complete (and reversiblein normal ACSF) blockade of middle layer responses during low calcium perfusion. For two of those slices, a short latency (3-4.5 ms) spike-like field potential remained (i.e., small and narrow) but only in the lower layers. These results suggest that the majority of the MG-evoked extracellular responses in the primary slice are synaptically mediated. The residual responses that sometimes remain in the lower layers likely result from direct activation of either afferent fibers or antidromically activated cells.

INTRACELLULAR RECORDINGS IN LAYERS 3-4 OF THE PRIMARY SLICE REVEAL CONSISTENT EARLY RESPONSE AND MORE IRREGULAR LATE POLYSYNAPTIC RESPONSE. Given the predominant middle layer CSD sinks and the known projection to this same region from the MGv, it seemed likely that major monosynaptic thalamocortical responses in the primary slice occur at the foci of the fast sinks in the middle cortical layers. Thus as a first step in the intracellular investigation of the thalamocortical responses, it seemed reasonable to direct recordings near those sinks. To do this, the auditory cortex was first mapped with an extracellular electrode to find the "focus" of the MG-evoked response in layers 3-4 (as described for the laminar recordings), then the intracellular recordings were conducted at or near that focus (within ~250 µM). Sixty-six cells, located between 30 and 50% of the distance from the pia to white matter, were recorded from 24 primary slices. Mean values of passive membrane properties (±SE) were as follows: resting potential = 69.3 ± 0.9 mV, input resistance = 46.8 ± 2.1 M, spike threshold (current) = 0.39 ± 0.03 nA, spike threshold (membrane potential) = 48.7 ± 0.5 mV, spike height = 64.1 ± 0.7 mV, spike width (at half-amplitude) = 0.87 ± 0.03 ms. Three cells had categorically narrower spikes (range: 0.28-0.44 ms) than the other 63 (range 0.64-2.21 ms). They also fired at high rates (100 Hz for hundreds of milliseconds during depolarizing current injection) and had deep/fast afterhyperpolarizations (AHPs), consistent with the fast spiking cell type (Connors and Gutnick 1990; McCormick et al. 1985; Porter et al. 2001). The majority of cells with wider spikes displayed strong spike frequency adaptation and had more shallow/gradually developing AHPs, consistent with the regular spiking cell type (Connors and Gutnick 1990; McCormick et al. 1985; Porter et al. 2001). We observed no obvious differences in synaptic responses between cell types and will report the data together.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Early and late responses of cells in layers 3-4 of the primary slice. A: responses of example cell to increasing intensities (in µA: 12, 25, 50, 100, 200). Each trace is average of 8-10 responses. Interstimulus interval (ISI) = 10 s. Note increases in slopes and amplitudes with increasing intensities. The apparent differences in spike thresholds are artifacts of averaging. Inset: 4 individual responses for 200-µA stimulus (2 with early and late responses, 2 with early responses only). Note that failure of late response results in smooth decay phase. Spikes are truncated. B: average early response amplitudes as a function of intensity. The "n" represent the number of cells for each intensity. Note the graded increases in peak amplitudes with increasing intensities. C: effects of "high divalent ACSF" (see METHODS) on late responses. Top: intracellular (same cell as in A), bottom: simultaneous middle layer field potential. Stimulus intensity = 200 µA, ISI = 30 s. Average of 10 trials. Note that average control late response at this slower stimulus rate is larger than average in A. Intracellular and extracellular late responses are strongly suppressed by high divalent ACSF, but the early response peaks are similar to control amplitudes. Explained further in RESULTS.

EARLY MG-EVOKED RESPONSE IN ACX CONTAINS FAST AMPA AND SLOWER NMDA EPSPS. Within the early response, there were multiple components that could be dissociated physiologically and pharmacologically. Figure 5A illustrates this for a cell in layer 4. The MG was stimulated at 0.1 Hz, which largely exhausted the late response discussed in the preceding text (not shown: note the difference in time scale relative to Fig. 4), and the remaining early response was recorded while the steady-state membrane potential of the neuron was varied with intracellular current injection. Because of unique voltage dependencies, the different subcomponents of the early response were most distinct at depolarized steady-state potentials (Fig. 5A, top). Following an MG stimulus in control solution (Fig. 5A1), there was an initial depolarization that peaked quickly (labeled fast EPSP), then a sharp negative deflection to below the baseline membrane potential (IPSP; discussed in the next 2 sections), and finally a second depolarizing peak (slow EPSP). Both the fast and slow EPSPs could trigger action potentials when the cell was "held" a few millivolts below threshold (shown for slow EPSP). The slow EPSP had clear nonconventional voltage dependence, being more prominent at depolarized steady-state potentials, suggesting the involvement of NMDA receptors.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Differential effects of glutamate receptor antagonists on the 3 components of the primary slice early response: fast excitatory postsynaptic potential (EPSP), fast inhibitory PSP (IPSP), slow EPSP. A1: example middle layer cell showing 3 components of early response. Resting potential = 68 mV. Steady-state potentials adjusted by injecting intracellular current (in nA, from bottom trace to top: 0.2, 0, 0.2, 0.4). MG stimulus time indicated by arrow. Stimulus intensity = 150 µA. Response components are labeled at most depolarized steady-state level. Traces at 3 most hyperpolarized steady-state potentials are averages of 7-10 responses. Individual responses presented for depolarized potential to illustrate trials with and without action potentials (AP) initiated by slow EPSP. For other trials, the fast EPSP could induce a spike. Scale bars apply to all intracellular traces (A, 1-3). A2: 20 µM 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX) blocked the fast EPSP and fast IPSP, leaving a slow EPSP with nonconventional voltage dependence. A3: the combination of 20 µM CNQX + 50 µM DL-2-amino-5-phosphonopentanoic acid (APV) blocked all the MG evoked responses for the cell (the slow EPSP recovered after removal of APV, not shown). B: field potential from same slice as in A (simultaneous, layer 3-4). CNQX blocked the large fast potential, leaving a smaller slow potential (B2). Addition of APV (CNQX + APV) blocked the remaining slow potential (B3), which recovered on subsequent removal of APV (B4, CNQX). A large portion of the fast potential recovered during 2.5-h rinse in normal ACSF (B4, ACSF). C: group effects of glutamate antagonists on fast EPSP (1), slow EPSP (2), and fast and slow field potentials (3; 9 cells, 9 field potentials). Fast EPSPs and field potentials were measured at latency corresponding to peak (initial 20 ms) in control conditions. Slow EPSPs and field potentials were measured at latency corresponding to peak of response during CNQX (at depolarized steady-state potentials for intracellular: the most depolarized value that was still sub-threshold for spiking was used). While the fast EPSP was nearly abolished by CNQX, the slow EPSP was only moderately suppressed; the latter effect was weaker for depolarized steady-state potentials than at resting potentials. Addition of APV reversibly blocked the slow EPSP (completely) even at depolarized potentials. The fast and slow group field potentials had similar effects as the corresponding EPSPs plus partial recovery in normal ACSF.

NATURE OF THE FAST IPSP. The cell in Fig. 5 expressed a clear IPSP in control solution. For example, while both the fast and slow EPSPs could trigger spikes, there were never action potentials during the IPSP, even when strong depolarizing current was injected into the cell to produce "spontaneous" action potentials (not shown). The IPSP had distinct voltage dependence, being more prominent at depolarized steady-state membrane potentials, and reversing at approximately 62 mV (Fig. 5A1). This data, together with the fast time-course, suggest a GABA_A receptor-mediated mechanism (Avoli 1986; Connors et al. 1988; Cox et al. 1992; Hefti and Smith 2000). Note that the IPSP was blocked during CNQX application, consistent with the interpretation that glutamatergic synapses drive the interneurons responsible for the IPSP (see DISCUSSION).

View larger version (28K): [in this window] [in a new window]   Fig. 6. Differences in responses evoked by MG vs. on-beam stimuli: IPSPs and laminar profiles. A: intracellular responses evoked by MG stimuli (100 µA) and on-beam stimuli (60 µA) for resting potentials (MG, 73 mV; on beam, 71 mV) and depolarized steady-state potentials (48 mV for both, using 1.0 nA of intracellular current). Each trace represents average of 7-10 responses (response with spikes excluded). Note stronger IPSP for on-beam stimulation, despite similar initial EPSP slopes for on-beam and MG stimulation. B: example of on-beam-evoked laminar field potential and CSD profile (gray scale derived from traces). The CSD profile is compared with an MG-evoked CSD profile from same slice. Conventions and procedures as in Fig. 3. Note the large on-beam-evoked CSD sinks in the supragranular and infragranular layers, whereas the large MG evoked sink is in the middle layers as usual. See RESULTS for detailed explanation of IPSP and laminar comparison.

MG-EVOKED RESPONSE DIFFERS FROM RESPONSE PRODUCED BY CONVENTIONAL COLUMNAR (ON BEAM) STIMULATION. Aside from recent studies with intact thalamocortical slices (see INTRODUCTION), in vitro studies have generally employed stimulation of layer 6, or the white matter just below layer 6, in an attempt to activate thalamocortical axons projecting to cells recorded in the cortical column above (discussed in Agmon and Connors 1991; Kenan-Vaknin and Teyler 1994; Kirkwood and Bear 1994). This conventional white matter/layer 6 stimulation will be referred to as "on-beam" stimulation. Here we compared responses in primary slices evoked by MG and on-beam stimulation and found clear differences between them relating to IPSP strengths and CSD distributions. These two results will each be described in turn.

TRACT TRACING IN THE PRIMARY SLICE: ANATOMICAL CONNECTIONS BETWEEN THE MGV AND ACX. To anatomically investigate the thalamocortical pathway in the primary slice, we performed tract tracing studies using the lipophilic dye Di-I. Following physiological recording, slices were fixed, then a particle of Di-I (50 µM diam) was placed either in the MG near the stimulation site (n = 8 slices) or in the ACx near the focus of the middle layer response (n = 5 slices). The dye was allowed to diffuse along the pathway(s) for 1-2 mo (30-38°C), then the slices were re-sectioned into 50-µM-thick segments and imaged using conventional epifluorescence microscopy (see METHODS).

View larger version (98K): [in this window] [in a new window]   Fig. 7. Anterograde and retrograde 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Di-I) tracing along the thalamocortical pathway in the primary slice. Orientation for all panels: anterior toward left, lateral toward bottom. The approximate positions of the images in A1 and B1 are indicated by the rectangle in Fig. 1D. A1: low-magnification image of MG, ACx, and surrounding structures, following Di-I application to the MG (*). - - -, a region that includes ACx and parts of striatum and is expanded in A2. The white arrow indicates a region of increased labeling density, approximately where the thalamocortical pathway crosses the reticular thalamic nucleus. A2: medium-magnification image showing the pattern of cortical labeling. In this section, labeling mostly involves anterograde-filled processes with many branches in layers 3-4. Other sections from same slice also had retrograde labeling of deep layer pyramidal cells. A3: high-magnification image from same section showing plexus of processes in layers 3-4. B1: low-magnification image following Di-I application to ACx (*). Orientation similar to A1. B2: part of MG outlined by dashed box in B1 is shown at higher magnification, illustrating 2 retrograde-filled MG somata. B3: image from adjacent section of same slice showing more MG somata. Details of the primary slice tracing presented in Tract tracing in the primary slice: anatomical connections between the MGv and ACx.

Shell slice

ANATOMICAL FEATURES OF THE SHELL SLICE. The shell slice was located ventral to the primary slice (Fig. 1). Gross anatomical observations (e.g., Fig. 1, C and E) and knowledge of the ventral boundaries of the primary slice (see preceding text) indicate that the thalamic portions of the shell slice are located below the MG proper. This region contains nonprimary or "shell" auditory thalamic subdivisions (e.g., the PPD and PIN; Figs. 1A and 2A) and more ventral nonauditory structures (e.g., substantia nigra, cerebral peduncle; Figs. 1A and 2A). Similar observations and reasoning suggest that the shell slice may contain portions of the "belt" auditory cortical area, ventral to primary ACx.

LAMINAR RESPONSE PROFILE OF SHELL SLICE IS DOMINATED BY A SURFACE CSD SINK. The dominant evoked response in the shell slice, to stimulation of the region ventral to the MG (indicated by arrow in Fig. 1E; hereafter referred to as the "shell region"), was a strong CSD sink on the surface of cortical layer 1 (Fig. 8). Generally, the field potential at the position of the sink was predominantly negative in polarity and had a similar time course as the sink. In contrast, the field potentials in the middle and lower layers were nearly always biphasic with a fast negative phase that preceded the surface sink and a slower positive phase having a similar latency as the surface sink. The example in Fig. 8A illustrates these and other characteristics of the shell slice laminar response (conventions and methodology as in primary slice experiments: Fig. 3). In addition to the surface sink, there was also a CSD source immediately below the sink (150 µM depth; Fig. 8A1). The gray-scale representation of this CSD profile is drawn to the right of traces and in the group plot (Fig. 8B).

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