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The Journal of Neurophysiology Vol. 87 No. 1 January 2002, pp. 361-384
Copyright ©2002 by the American Physiological Society
Department of Neurobiology and Behavior, University of California, Irvine, California 92697
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ABSTRACT |
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Cruikshank, Scott J.,
Heather J. Rose, and
Raju Metherate.
Auditory Thalamocortical Synaptic Transmission In Vitro.
J. Neurophysiol. 87: 361-384, 2002.
To
facilitate an understanding of auditory thalamocortical mechanisms, we
have developed a mouse brain-slice preparation with a functional
connection between the ventral division of the medial geniculate (MGv)
and the primary auditory cortex (ACx). Here we present the basic
characteristics of the slice in terms of physiology (intracellular and
extracellular recordings, including current source density analysis),
pharmacology (including glutamate receptor involvement), and anatomy
(gross anatomy, Nissl, parvalbumin immunocytochemistry, and tract
tracing with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). Thalamocortical transmission in this preparation (the
"primary" slice) involves both
-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid/kainate and
N-methyl-D-aspartate-type glutamate receptors that appear to mediate monosynaptic inputs to layers 3-4 of ACx. MGv
stimulation also initiates disynaptic inhibitory postsynaptic potentials and longer-duration intracortical, polysynaptic activity. Important differences between responses elicited by MGv versus conventional columnar ("on-beam") stimulation emphasize the
necessity of thalamic activation to infer thalamocortical mechanisms.
We also introduce a second slice preparation, the "shell" slice, obtained from the brain region immediately ventral to the primary slice, that may contain a nonprimary thalamocortical pathway to temporal cortex. In the shell slice, stimulation of the thalamus or the
region immediately ventral to it appears to produce fast activation of
synapses in cortical layer 1 followed by robust intracortical
polysynaptic activity. The layer 1 responses may result from
orthodromic activation of nonprimary thalamocortical pathways; however,
a plausible alternative could involve antidromic activation of
corticotectal neurons and their layer 1 collaterals. The primary and
shell slices will provide useful tools to investigate mechanisms of
information processing in the ACx.
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INTRODUCTION |
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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
).
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METHODS |
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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).
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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, H2O
usually 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).
For the most part, we adopted the auditory forebrain divisions of the Franklin and Paxinos (1997)| |
RESULTS |
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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
).
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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.
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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 reversible
in 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.
9.5 mV. When such cells were depolarized (with steady intracellular
current) to a few millivolts below spike threshold, the MG-evoked early
responses could usually trigger action potentials, indicating the
presence of EPSPs; however, these early EPSPs did not generally elicit
spikes from the resting potential.
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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.
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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 GABAA 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).
64 mV,
consistent with GABAA), and 5 cells displayed no
apparent IPSPs. Minimum fast IPSP latencies range from 4.8 to 10.8 ms
(mean = 6.8 ± 0.4 ms). The latencies between the onsets of
the EPSPs and IPSPs for these responses ranged from 1.4 to 6.2 ms
(mean = 2.8 ± 0.3 ms).
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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.
71 to
73 mV), the IPSPs were
positive going and added with the EPSPs to produce a net depolarizing
PSP. Notice that the initial slopes were approximately equal, and in
this case, the MG-evoked depolarization was actually larger than the
on-beam response. Despite the apparently equal or greater synaptic
drive, when the cell was depolarized with intracellular current
injection (to about
48 mV with +1.0 nA), the MG stimulus evoked a
weaker IPSP than the on-beam stimulus. While the large on-beam IPSP
hyperpolarized the cell several millivolts below the steady-state
membrane potential, the MG-evoked IPSP was weak, only cutting a small
notch in the EPSP (~1 mV deflection), and never reaching down to the
steady-state potential.
The IPSP differences indicate that on-beam and MG stimuli evoke middle
layer responses with different excitatory-inhibitory balances,
suggesting that they activate different intracortical cell groups. To
examine this on a larger spatial scale, we compared the laminar
profiles produced by MG versus on-beam stimulation within six primary
slices (Fig. 6B). Stimulus intensities at the two sites were
adjusted in an attempt to equalize their respective layer 4 field
responses, and the on-beam and MG stimuli were interleaved.
The thalamocortical profiles of the six slices were among those
presented in Fig. 3, therefore the MG-evoked CSDs were all fairly
typical, with largest sinks in the middle layers (30-50% of distance
between pia and white matter). In contrast, the laminar profiles evoked
by on-beam stimulation were more variable from slice to slice, with
maximum sinks at a variety of positions, including supragranular and
infragranular layers. The cortical depths of the largest on-beam sinks,
expressed as a percentage of the distance from the pia to white matter,
were: 18, 20, 30, 44, and 70%. The sixth slice had no clear cortical
sink produced by the on-beam stimulus. In 5/6 slices, the positions of
the largest MG and on-beam evoked sinks mismatched by a minimum of two
recording positions (
250 µM; e.g., Fig. 6B). The
differences in laminar profiles between MG and on-beam stimuli further
indicate that the two stimulation sites do not generally produce the
same patterns of cortical activation.
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).
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75% of the distance from pia to white
matter) or around the layer 5/6 border. Finally, in addition to the
example, one other slice had clear axons in layer 1. The density of
this labeling was considerably higher than in the example and included
many axons oriented parallel with the cortical surface.
Of the five primary slices with Di-I particles placed in the ACx, 4/5
had retrograde labeling of cell bodies in the MG. Figure 7B
presents an example. Notice that pathway appears to be interrupted (Fig. 7B1); the middle region of the pathway is actually
labeled in more ventral sections of the slice (not shown). This was a common feature and indicates that the thalamocortical pathway is curved
in the dorsal-ventral plane. It is most ventral in the middle region,
near the reticular thalamic nucleus, and curves dorsally toward both
extremities (near the MG and ACx; see Fig. 2C for
orientation). Consistent with this, labeled cell bodies in MG were
always located near the dorsal surface of the slice. It appears that
the ventral dip in the middle of the pathway is necessary so that the
thalamocortical fibers can pass under the fimbria; this can be best
appreciated by examining the structures present at different
dorsal-ventral levels in the parvalbumin material (Fig. 2C).
Taken together, the anterograde and retrograde labeling indicates that
the primary slice contains anatomically connected axonal pathways
linking the MG and ACx. Furthermore the projections from the MG seem to
end predominantly in cortical layers 3-4, although axons are also
usually present in the lower layers, and occasionally in layer 1. These
three projections could contribute, respectively, to the middle layer,
lower layer, and surface CSD sinks characterized previously
(LAMINAR RESPONSE PROFILE OF PRIMARY SLICE IS DOMINATED BY MIDDLE
LAYER CSD SINK, Fig. 3).
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.
Direct histological information was also obtained following recordings from shell slices (Nissl stain: n = 17; PV-immunohistochemistry: n = 1). For 12/18 shell slices in which thalamic regions were examined, the top 50-100 µM appeared to be within the PPD; for the remaining 6/18, the tops were located more ventrally, in the cerebral peduncle or substantia nigra (for orientation, see Fig. 2, A and B). For 5/12 shell slices in which cortices were examined (6 removed because of low-quality histology), the temporal cortices were well laminated on the top 50-100 µM. For the other 7/12, lamination was poor (even in the most dorsal sections), indicating localization ventral to primary ACx. The bottoms of the shell slices always appeared to be located below the PPD and 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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