Department of Anatomy and The Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin 53706-1532
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ABSTRACT |
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Bartlett, Edward L. and
Philip H. Smith.
Anatomic, intrinsic, and synaptic properties of dorsal and ventral
division neurons in rat medial geniculate body. Presently little is known about what basic synaptic and cellular mechanisms are
employed by thalamocortical neurons in the two main divisions of the
auditory thalamus to elicit their distinct responses to sound. Using
intracellular recording and labeling methods, we characterized anatomic
features, membrane properties, and synaptic inputs of thalamocortical
neurons in the dorsal (MGD) and ventral (MGV) divisions in brain slices
of rat medial geniculate body. Quantitative analysis of dendritic
morphology demonstrated that tufted neurons in both divisions had
shorter dendrites, smaller dendritic tree areas, more profuse
branching, and a greater dendritic polarization compared with stellate
neurons, which were only found in MGD. Tufted neuron dendritic
polarization was not as strong or consistent as earlier Golgi studies
suggested. MGV and MGD cells had similar intrinsic properties except
for an increased prevalence of a depolarizing sag potential in MGV
neurons. The sag was the only intrinsic property correlated with cell
morphology, seen only in tufted neurons in either division. Many MGV
and MGD neurons received excitatory and inhibitory inferior colliculus (IC) inputs (designated IN/EX or EX/IN depending on
excitation/inhibition sequence). However, a significant number only
received excitatory inputs (EX/O) and a few only inhibitory (IN/O).
Both MGV and MGD cells displayed similar proportions of response
combinations, but suprathreshold EX/O responses only were observed in
tufted neurons. Excitatory and inhibitory postsynaptic potentials
(EPSPs and IPSPs) had multiple distinguishable amplitude levels
implying convergence. Excitatory inputs activated
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and
N-methyl-D-aspartate (NMDA) receptors the
relative contributions of which were variable. For IN/EX cells with
suprathreshold inputs, first-spike timing was independent of membrane
potential unlike that of EX/O cells. Stimulation of corticothalamic
(CT) and thalamic reticular nucleus (TRN) axons evoked a
GABAA IPSP, EPSP, GABAB IPSP
sequence in most neurons with both morphologies in both divisions. TRN
IPSPs and CT EPSPs were graded in amplitude, again suggesting
convergence. CT inputs activated AMPA and NMDA receptors. The NMDA
component of both IC and CT inputs had an unusual voltage dependence
with a detectable DL-2-amino-5-phosphonovaleric acid-sensitive component even below
70 mV. First-spike latencies of
CT evoked action potentials were sensitive to membrane potential regardless of whether the TRN IPSP was present. Overall, our in vitro
data indicate that reported regional differences in the in vivo
responses of MGV and MGD cells to auditory stimuli are not well
correlated with major differences in intrinsic membrane features or
synaptic responses between cell types.
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INTRODUCTION |
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A set of auditory thalamic nuclei, known collectively as the
medial geniculate body (MGB), receives, modifies, and transfers sensory
information largely to specific regions of the cortex. In the two major
MGB subdivisions, the ventral or lemniscal (MGV) and the dorsal or
extralemniscal (MGD) divisions, cells often respond differently to
acoustic stimuli (see Clarey et al. 1992 for a recent
review). Extracellular recordings, made primarily in anesthetized cat
but also from rodent and monkey, indicate that MGV thalamocortical
neurons are arranged tonotopically, with many showing narrow frequency
tuning and a consistent short-latency response to repeated sounds. MGD
neurons, in contrast, are not tonotopically arranged and can display
broad frequency tuning, more variable response latencies, and rapid
habituation to repeated sounds (Aitkin and Webster 1971
,
1972
; Allon et al. 1981
; Bordi and Ledoux
1994
; Calford 1983
; Calford and Webster
1981
; Imig and Morel 1985
). Currently it is not
known how factors like membrane properties, dendritic architecture, or
synaptic input features contribute to these distinct response
differences. Moreover because some cells in one division may respond
differently to auditory stimuli when compared with others in that same
division (Aitkin and Webster 1971
; Bordi and
Ledoux 1994
), it is not known whether these factors can be
correlated with a particular response type.
Besides differing in response features, MGB neurons also differ
morphologically. Cat and rat Golgi studies indicate that two main
classes of thalamocortical neurons can be distinguished qualitatively in the two major subdivisions (Clerici and Coleman 1990;
Clerici et al. 1990
; Morest 1964
,
1965a
,b
; Winer 1984
). In MGV, "tufted" neurons have bushy secondary dendrites that result from lower-order dendrites giving rise to numerous branches in close proximity. In cat
and rat, these cells are reported to have a restricted or
"bitufted" domain polarized along the long axis of the cell body
and parallel to the ascending fibers of the brachium of the inferior
colliculus (IC). In the MGD, many neurons have "stellate" morphology due to radially extending, lower-order dendrites typically generating two higher-order dendrites that diverge to form a star-like configuration (Clerici and Coleman 1990
; Clerici
et al. 1990
; Morest 1964
, 1965b
; Winer
and Morest 1983
, 1984
). Some MGD neurons can have tufted-like
morphology but do not display oriented dendritic trees (Clerici
et al. 1990
; Winer 1992
).
Except for an early study (Nelson and Erulkar 1963),
intracellular response features of MGB neurons in vivo are unavailable, but there are several reports of intracellular recordings in slices. The surprising conclusion of the pioneer work of Jahnsen and
Llinas (1984a
,b
) was that thalamocortical cells, including four
from the MGB, displayed similar electrophysiological features
regardless of morphological differences or thalamic location. This
finding indicated that intrinsic membrane features were probably not
related to cell to cell response differences to natural sensory stimuli in vivo. Recent work (Hu 1995
; Hu et al.
1994
; Senatorov et al. 1997
) has
challenged this perception in the auditory thalamus by proposing that
rat MGV and MGD cells may have different intrinsic features that could
influence their in vivo response. A hyperpolarization-activated cation
current (Ih) found in most MGV neurons could
produce a depolarizing "sag" that was not evident in most MGD
neurons (Hu 1995
). It was suggested that
Ih produced more depolarized resting potentials
in MGV neurons, putting them in the "single spike," or tonic,
mode. In contrast, MGD neurons without Ih were
more hyperpolarized and in the Ca2+-dependent "burst
mode" of thalamic firing (Hu 1995
; Hu et al. 1994
; Jahnsen and Llinas 1984a
). Given that a
single thalamic neurons can switch between firing modes and that
synaptic responses of cells in the two firing modes would differ when
presented with the same input, control of resting potential is a
potentially important feature in determining responses to sensory
stimuli. However, the cells were selected populations from restricted
regions of the MGV and MGD and only a few cells were labeled
intracellularly (Hu 1995
; Hu et al.
1994
).
In addition to anatomic and extracellular response differences, the
origin of afferent inputs to MGV and MGD cells differ as well. Most
ascending MGV afferents arise from cells in the laminated,
tonotopically organized central nucleus of the IC (Andersen et
al. 1980; Calford and Aitkin 1983
; Kudo
and Niimi 1978
, 1980
; LeDoux et al.
1985
; Rouiller and de Ribaupierre 1985
).
Ascending auditory inputs to MGD arise from other regions of the IC and the lateral tegmental system (see Winer 1992
) and show
no obvious laminar distribution. Recent evidence in the cat and rat
(Peruzzi et al. 1997
; St. Marie et al.
1997
; Winer et al. 1996
) indicates that some
collicular inputs to both regions are GABAergic. The other major
afferent inputs to MGB are excitatory inputs originating from auditory
cortex and GABAergic inputs from the thalamic reticular nucleus (TRN)
(Jones 1975
; Jones and Powell 1969a
,b
;
Jones and Rockel 1971
; Montero 1983
;
Rouiller et al. 1985
). TRN cells are organized
topographically and project to both dorsal and ventral divisions
(Crabtree 1998
). Cortical inputs to MGB cells are mainly from layer VI pyramidal cells in primary auditory cortex (to MGV) or
nonprimary auditory cortex (to MGD) the axons of which give rise to
small, excitatory terminals thought to synapse on distal dendrites
(Bajo et al. 1995
; Ojima 1994
;
Rouiller and Welker 1991
). Some evidence exists
regarding the influence of these inputs on extracellular MGB cell
responses in vivo (Ryugo and Weinberger 1976
; Yan
and Suga 1996
; Zhang et al. 1997
), but
there are no data on their intracellular features.
In this study, we sought to clarify the relationship among cell location, cell morphology, intrinsic properties, and synaptic properties of MGV and MGD neurons. We recorded intracellularly from cells in both divisions of the rat MGB using brain slices and labeled these physiologically characterized cells. To correlate the membrane features and synaptic properties with morphology, the morphological type of a number of neurons was established using more quantitative methods. We then categorized and compared the basic membrane features of these different cell types in the two major divisions and compared the influences of ascending and descending synaptic inputs. Our analysis suggests that, in the rat slice, neurons in both regions with either cell morphology can have similar resting potentials, firing modes, proportion of response patterns to IC input (with one exception), and responses to corticothalamic and thalamic reticular nucleus stimulation. Overall our results imply that the marked contrast between the in vivo responses of MGV versus MGD neurons may be due to differences in the afferent response patterns and the selectivity of each afferent input to auditory stimuli.
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METHODS |
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Intracellular recording
Methods for intracellular recording, stimulation, and labeling
of MGB neurons are described elsewhere (Peruzzi et al.
1997; Smith 1992
). All methods were approved by
the University of Wisconsin Institutional Animal Care and Use
Committee. Animals were maintained in an American Associations for
Accreditation of Laboratory Animal Care-approved facility. Briefly, 3- to 6-wk-old hooded rats were anesthetized deeply (chloral hydrate, 70 mg/ml, 1-1.5 ml ip, or pentobarbital sodium, 10 mg/ml, 0.5 ml ip) and
perfused transcardially with chilled, oxygenated sucrose saline
(described in the following text). Four-hundred- to 500-µm slices in
the desired plane of section containing the MGB were then made as
described previously an placed in a holding chamber with oxygenated
room temperature saline (Peruzzi et al. 1997
;
Smith 1992
). After equilibrating, a slice was
transferred to the recording chamber and perfused with oxygenated
normal saline at 33-34°C containing the following (in mM): 124 NaCl,
5 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 26 NaHCO3, and 10 glucose. Sucrose
saline contained sucrose instead of NaCl (Aghajanian and
Rasmussen 1989
). In experiments that assessed the effect of
extracellular [K+] on membrane properties, we used a
bathing solution which contained the following (in mM): 125 NaCl, 1.8 KCl, 1.2 KH2PO4, 1.8 CaCl2, 1 MgSO4, 26 NaHCO3, and 10 glucose. This saline
had a final [K+] similar to that used in previous in
vitro MGB studies (Hu 1995
; Hu et al.
1994
).
In horizontal and sagittal slices, one stimulating electrode was
positioned in the thalamic radiations (see Paxinos and Watson 1986, plates 84 and 103) to excite corticothalamic and thalamic reticular nucleus fibers. The other was placed in the brachium of the
inferior colliculus to excite IC axons (see Peruzzi et al.
1997
, Fig. 5).
Bicuculline methiodide, 2-hydroxysaclofen, DL-2-amino-5-phosphonovaleric acid (APV), 6,7-dinitroquinoxaline (DNQX), SR-95531 (Research Biochemicals International, Natick, MA), and CGP35348 (Ciba-Geigy, West Caldwell, NJ) were all mixed with artificial cerebrospinal fluid (ACSF) to the stated concentrations the day of the experiment and bath applied.
Glass microelectrodes (80-200 M) filled with 2 M potassium acetate
containing 2% Neurobiotin were used to record intracellularly. After
recording and injection, the slice was removed from the recording
chamber and fixed. Neurobiotin-labeled MGB neurons were prepared and
reacted as previously described (Peruzzi et al. 1997
; Smith 1992
). Two-dimensional camera lucida drawings of
injected cells were made at ×1,250. Cell body location relative to the MGB divisions was determined using the Paxinos and Watson
(1986)
atlas and the cytoarchitectural study of the rat MGB by
Clerici and Coleman (1990)
.
Data analysis
PHYSIOLOGY.
Intracellular current and voltage records were digitized and sampled by
a PC using software developed here. The difference between the voltage
measured extracellularly in saline and intracellularly during recording
was taken as the resting membrane potential. Input resistance was taken
as the slope of the linear portion of the current-voltage plot near the
resting potential or calculated from the maximum voltage deflection to
a 0.1-nA current pulse. The I-V response was
"rectified" if voltage responses to equal-amplitude, opposite-polarity currents differed by
25%. If the maximum voltage deflection to a 200-to 300-ms hyperpolarizing current differed from
that during the last 25 ms by
2 mV, the cell was said to have a
"sag." Cells were in burst mode at rest if depolarization evoked
a large long-lasting depolarizing event often crested by multiple
high-frequency action potentials (see Fig. 3B). The burst depolarization depends on extracellular Ca2+ (not shown)
and has been seen in MGB and elsewhere in the thalamus (Hu
1995
; Jahnsen and Llinas 1984a
,b
;
Tennigkeit et al. 1996
).
ANATOMY. The somas and primary dendrites of labeled neurons were drawn at ×1,250, and soma areas were calculated using Neurolucida software (Microbrightfield, Colchester, VT). Only well-labeled somas with no signs of electrode induced deformation were used. Dendritic arbor area was measured using the same software by drawing a line between the ends of adjacent dendrites to create an area enclosing the entire dendritic tree, then measuring the enclosed area. Dendritic projection distance from the soma was calculated by measuring the length of the dendrite from the soma. Scaled circles representing 50 or 100 µm were used to count the number of dendrites extending beyond 50 or 100 µm, and the sum was termed the number of "dendritic intersections" at these radii. Each circle was centered around the soma center and divided into eight 45° sectors, and the number of dendrites intersecting each sector at 50 and 100 µm was counted. Also the number of sectors (directions) with one or more dendrites intersecting at 50 and 100 µm was counted. To estimate directional uniformity of dendritic projections, we devised a simple measurement termed the orientation ratio. This ratio is the sum of the number of dendritic intersections in the four sectors with the most intersections divided by the sum of the number in the four sectors with the fewest. If the sum of the four sectors with the fewest intersections totaled zero, it was set to one to avoid dividing by zero. A dendritic tree with similar numbers of dendrites projecting in each direction would have an orientation ratio close to one, whereas a tree with dendrites preferentially projecting in a small number of directions would have a large orientation ratio.
Analysis of the fully reconstructed dendritic trees of 23 neurons at ×1,250 demonstrated that stellate neurons had significantly longer dendrites than tufted neurons (Table 1, stellate range: 179-231 µm, tufted range: 123-176 µm). An additional 30 neurons were drawn at a lower magnification, and their dendritic lengths were measured. Those whose three longest dendrites averaged < 160 µm were called tufted and those that averaged > 175 µm were called stellate. A few of these neurons whose dendrites averaged between 160 and 175 µm could not be classified solely by dendritic length.
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RESULTS |
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The database for this study consists of 106 MGB neurons that were
recorded intracellularly. Certain aspects of some of the neurons have
been considered in a previous study (Peruzzi et al. 1997).
Qualitative descriptions of MGV and MGD neurons
Figures 1 and
2 illustrate several examples of
labeled thalamocortical cells from each MGB division. Cell somas had no
consistent shape or size, regardless of MGB subdivision, plane of
section, or dendritic tree appearance. The axon arose from the cell
body or proximally from a primary dendrite (Figs. 1 and 2,
). No axon collaterals ever were observed within the MGB.
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In the ventral division, dendritic trees were fairly dense and
intertwined, resembling previous descriptions of dendritic tufts
(Clerici et al. 1990; Winer 1992
). MGV
neurons sometimes displayed an oriented dendritic tree. For example,
cells 1, 2, 6, and 7 in Fig. 1 demonstrate fairly
clear orientation in the horizontal (cells 1, 6, and
7) and sagittal (cell 2) planes. Other cells,
including cells drawn in the coronal (cells 5 and
8) and horizontal planes (cell 3), did not
display clear dendritic orientation but rather projected in all
directions in the plane. The primary dendrites of MGV neurons were
fairly smooth, whereas higher-order dendrites had irregular surfaces
with noticeable protrusions but very few dendritic spines.
Some MGD neurons had tufted dendritic trees resembling those in the
ventral division, with a comparable number of primary dendrites that
branched profusely and showed varying amounts of orientation (Fig. 2,
cells 2-5). Another group had larger dendritic trees that
did not branch as profusely and extended in all directions in the
horizontal or coronal planes, conforming to earlier descriptions of MGD
cells with "stellate" morphology (Clerici et al.
1990; Winer 1992
) (Fig. 2, cells 1, and 6-8). However, it was often difficult to qualitatively
assess whether the features of these neurons, for example dendritic
length, branchiness of the dendritic trees close to the cell body, and
dendritic orientation, could distinguish stellate versus tufted
morphology. Therefore we made a series of measurements on the dendritic
arbors of several well-filled MGB neurons to see more objectively
whether two distinct dendritic morphologies are indeed found in MGB.
Quantitative analysis of somas and dendrites of MGB thalamocortical neurons
Measurements were made on 42 cell bodies, 19 from MGV and 23 from MGD. Table 1 shows that the measured soma areas were similar in dorsal and ventral divisions. Also compared in Table 1 are the dendritic characteristics of 10 well-labeled MGV neurons and 13 well-labeled MGD neurons. Ventral division neurons had a fairly small range of arbor areas (20,000-35,000 µm2). MGD neurons, by contrast, had two separate area ranges, with a total range of 19,000-81,000 µm2. One range of MGD areas overlapped with the MGV areas, whereas the other range was significantly larger and did not. Therefore we classified neurons with dendritic areas <40,000 µm2 as small area neurons and neurons with dendritic areas >40,000 µm2 as large area neurons and then analyzed the dendritic characteristics of the two groups. The dendritic areas of the small area MGD neurons were 27,800 µm2 on average, comparable with the average MGV area (Table 1), whereas those of the large area MGD neurons were 62,800 µm2. Measuring the three longest dendrites revealed that small area neurons in both divisions had similar dendrite lengths. Dendrites of MGD large area neurons were significantly longer (see Table 1), indicating that differences in dendritic tree area was at least partially due to a difference in dendrite length.
Grouping cells according to the area of their dendritic trees revealed other consistent differences between the two populations. The number of dendritic intersections (see METHODS) for small area neurons in either MGV or MGD was significantly larger than for large area neurons at 50 µm, but significantly smaller at 100 µm. This result indicates that small area arbors were more exuberant at 50 µm and that many small area neuron dendrites ended between 50 and 100 µm from the soma. When the number of sectors intersected (see METHODS) was measured, at 50 µm, small and large area dendrites extended almost omnidirectionally, with most neurons having dendrites intersecting seven or eight of eight possible sectors. Dendrites of large area neurons continued to project in all directions 100 µm from the soma, but dendrites of small area neurons on average projected through only six of eight possible sectors. Shorter dendrites and more "oriented" (see following text) dendritic tree characteristics of small area neurons probably contributed to the decrease in the number of sectors intersected at 100 µm. Orientation ratios (see METHODS) for large area neurons at 100 µm were significantly smaller (Table 1), i.e., large area neuron dendrites are less oriented. However, it should be noted that 6/17 small area neurons had ratios similar to large area neurons at 100 µm, supporting our qualitative observation that not all small area cells have strongly oriented dendritic arbors. Finally, we found that small area cell dendrites in both MGV and MGD tended to have all branching occur 20-60 µm from the soma, whereas large area dendritic branching occurred fairly evenly 20-120 µm from the soma. Examples of distal branchpoints are seen in all the large area arbors in Fig. 2 (cells 1, and 6-8). Based on these quantitative results, the small area neurons conform to qualitative descriptions of tufted neurons, whereas the dendritic characteristics of the large area neurons conform to descriptions of stellate neurons. Although the distribution of dendritic lengths of tufted (small area) and stellate (large area) neurons was nonoverlapping, individual tufted neurons could have nonoriented dendritic arbors, and individual stellate dendrites could have branching nearly as dense as typical tufted arbors.
Intrinsic properties of MGB neurons
The intrinsic properties of 31 MGV neurons and 25 MGD neurons were
analyzed in detail. As shown in Table 2,
the average resting membrane potential and average input resistance
were similar throughout the MGB regardless of location or cell type.
Suprathreshold depolarizing current pulses elicited burst firing in
about one-third of the MGV and MGD neurons, characterized by a large
hump crested by one or more action potentials in a high-frequency burst
(Fig. 3B). This response was
not confined to a particular cell type. Cells in the burst firing mode
had a resting potential of 69.8 ± 3.3 (SD) mV. All MGB
neurons could generate the burst after the offset of hyperpolarizing
current (a rebound burst, Fig. 3, A and B). The
average number and frequency of action potentials in the rebound burst
was similar throughout the MGB (Table 2). The resting potential of
bursting neurons was significantly more hyperpolarized than those
showing the tonic, or sustained, firing mode at rest. The tonic firing
mode, which is similar to the "single-spike" mode of previous
studies (Hu 1995
; Hu et al. 1994
), was
characterized by firing for all or most of the duration of the current
injection (Fig. 3D). Neurons in this firing mode had an
average resting potential of
61.7 ± 4.4 mV and could be either
morphology. Firing rate increased and first-spike latency decreased
with increasing current (Fig. 3D). About half of the neurons
in the tonic firing mode displayed noticeable spike rate adaptation.
Switching the membrane potential by injecting constant current into a
cell changed the firing mode of the neuron, indicating that the ability
to burst or fire tonically is possessed by all MGB neurons.
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In our normal saline, there was no segregation of cells, based on
anatomic type or location, into a particular firing mode. Because the
finding that MGV and MGD neurons have similar resting potentials
differs from previous reports comparing MGV and MGD neurons (Hu
1995; Hu et al. 1994
), extracellular
[K+] was changed to match values used in the previous
studies. Cells switched from our normal saline to one containing 3 mM
[K+] showed a consistent hyperpolarization of the
membrane potential (3-5 mV) in both dorsal and ventral divisions. This
manipulation could change a nonbursting cell into the burst firing mode
or lower the amount of current needed to generate a burst in an already bursting cell. However, the lower extracellular [K+] did
not divide MGB neurons into tonic-firing MGV and burst-firing MGD
populations. Also, because the preparation used in previous studies
restricted the region of MGB that could be sampled, neurons in the
rostral quarter and the medial third of the MGV and MGD were excluded.
When the remaining neurons were analyzed, the average resting membrane
potential and proportion of neurons bursting at rest were similar
between MGV and MGD. Finally, 15 MGD stellate and 12 MGV tufted neurons
were identified anatomically (see METHODS), and their
intrinsic properties were compared. Again, all measured properties were
similar with one exception. This exception was the prevalence of a
depolarizing "sag" during a hyperpolarizing current pulse in most
MGV neurons (Fig. 3A), which occurred only in a small number
of MGD neurons (Table 2). Of the four identified MGD cells showing the
sag, two (small sags in these neurons) had stellate morphology, and two
had tufted morphology. The I-V curve in Fig. 3C,
comparing the peak change in membrane potential (
) with the change
observed just before current offset (+) in an MGV neuron, reflects this
sag as well as an outward rectification. The sag is thought to reflect
activation of a Cs+-sensitive, mixed cation current
(Ih) (Hu 1995
; Tennigkeit
et al. 1996
). Also, a smaller voltage deflection was observed
for depolarizing compared with hyperpolarizing currents in most MGB neurons (Fig. 3, A and C). The rectification
probably reflected 4-aminopyridine-sensitive K+ currents
activated by membrane depolarization in MGB and lateral geniculate nucleus neurons (McCormick
1991
; Tennigkeit et al. 1996
, 1998
).
Another possible effect of K+ current activation
is the afterhyperpolarization (ahp) that often occurred after the
offset of depolarizing current and could last hundreds of milliseconds
(Table 2, Fig. 3B).
IC inputs to MGB neurons
We previously described direct excitatory and inhibitory IC inputs
to MGB neurons (Peruzzi et al. 1997). Over half of the tufted and stellate cells reported in this study (38/72) received both,
but there were variations. Figure 4
illustrates the four patterns of IC synaptic input seen when
colliculogeniculate axons were stimulated electrically in the brachium
of the inferior colliculus (BIC). These patterns were named based on
the presence of a GABAA IPSP and its latency relative to
the EPSP latency. MGB neurons with only excitatory IC input were called
EX/O. EX is for "excitatory" and O signifies a lack of an
apparent GABAA IPSP (Fig. 4, E and F). Neurons with only inhibitory IC input were named IN/O,
IN is for "inhibitory" IPSP and O signifies a lack of a driven
EPSP (Fig. 4, G and H). If a GABAA
IPSP preceded the EPSP, the response was called IN/EX (Fig. 4,
A and B), and if the EPSP preceded the IPSP, it
was called EX/IN (Fig. 4, C and D). Because
neurons with GABAA IPSPs almost always had a
GABAB component (43/44 of the IC inputs, 68/73 of the
descending inputs described in the following text), the
GABAB component was not included in the nomenclature. With
one exception (see following text), both tufted and stellate could show
any of the four patterns of synaptic inputs, and in this small sample
of cells, there was no obvious difference in the distribution of the
four patterns between the two cell types in the two MGB divisions
studied.
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When gradually increasing the shock strength applied to the BIC, the EPSPs and IPSPs often had multiple discernible amplitudes, implying that multiple IC fibers converge onto single MGB neurons. The number of excitatory inputs converging on a single MGB neuron was small, with two to four distinguishable amplitudes in most cases (3.0 ± 1.9) (Fig. 5, A and B). Multiple levels of excitation were noted for all response patterns. In most cases, the multiple peak amplitudes were not due to the addition of an increasing inhibition to a constant excitation because peak amplitude increased with stimulus intensity. Also, multiple peak amplitudes still were noted when the GABAA channel was blocked (not shown). Inhibitory IC inputs occasionally were isolated either by application of glutamate channel blockers APV and DNQX or by stimulation of only inhibitory fibers. Similar to the excitatory fibers, increasing the shock stimulus amplitude generated multiple IPSP amplitude levels (Fig. 5, C and D). In most cases, the GABAB IPSP had the same, or a slightly higher, stimulation threshold as the GABAA IPSP (Fig. 5, C and D). Thus more than one IC inhibitory input also converges onto MGB relay neurons. Generally, the number of discernible excitatory levels was independent of input class, but some of the EX/O neurons received a large, all-or-none EPSP. An example is shown in Fig. 4E. When the intensity of the stimulus was low, no response above baseline was seen. Increasing stimulus intensity slightly evoked a large suprathreshold EPSP. Although it is difficult to tell if subsequent smaller EPSPs were obscured, the evoked response size and duration did not change as stimulus intensity was increased further, suggesting that only one fiber was being stimulated.
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Neurons receiving each type of IC input were found in similar proportions in the dorsal and ventral divisions. Table 3 shows that there are approximately equal numbers of neurons receiving EX/O and IN/EX input, the two most common classes of input. However, only neurons with tufted morphology received a single, large, suprathreshold EPSP. There was a higher percentage of EX/IN neurons in the dorsal division (23%) than in the ventral division (7%), but the difference was not statistically significant. About 10% of the neurons in either subdivision received exclusively inhibitory IC input. It is possible that some cells exhibiting EX/O or IN/O patterns actually receive both excitatory and inhibitory inputs from the IC because a small number of EX/O responses also showed a long-duration IPSP (probably GABAB mediated), but the stimulus electrodes only excited the fiber type whose response was observed.
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The PSP latency was related to the type of IC input. For these monosynaptic inputs to MGB neurons, PSP latency provides a measure of the relative axonal conduction velocity of each input. The EPSP latencies of EX/O inputs were significantly shorter than the latencies of IN/EX EPSPs (Table 3) in both subdivisions. Bath application of the GABAA antagonists bicuculline or SR95531 to neurons with IN/EX or EX/IN IC inputs did not change the EPSP latency (Fig. 6,C and E). Early GABAA IN/EX IPSPs had similar latencies to the EX/O cell EPSPs throughout the MGB (Table 3), suggesting that axons giving rise to EX/O excitatory input and IN/EX inhibitory input had similar, relatively fast conduction velocities. MGV neurons with EX/IN inputs had GABAA IPSP latencies like IN/EX GABAA latencies, but their EPSP latencies were among the fastest observed. In contrast, MGD GABAA IPSPs in EX/IN cells were significantly longer than all other GABAA latencies, whereas the EPSP latencies for these neurons were significantly shorter than the IN/EX EPSP latencies. IN/O GABAA latencies were comparable with the GABAA latencies of IN/EX inputs. On the basis of these results, IC excitatory input can be subdivided into short-latency inputs, which are generally seen in EX/O or EX/IN categories, and long-latency inputs, which are generally in the IN/EX category. The inhibitory IC inputs also can be grouped according to latency. Shorter-latency IPSPs are associated with IN/EX, IN/O, and MGV EX/IN inputs, whereas longer-latency IPSPs occur in MGD EX/IN responses.
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Pharmacology of IC inputs
EPSPs for stellate and tufted neurons had both
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and
N-methyl-D-aspartate (NMDA) components. There
was considerable variation in how much each component contributed to
the EPSP. Like other dual component glutamatergic EPSPs, blocking AMPA
channels diminished the fast portion of the EPSP, resulting in a
reduced peak amplitude and a longer time to peak. Figure 6A
shows a MGV tufted neuron response that still has a large NMDA
component during application of the AMPA channel antagonist DNQX.
Blocking the AMPA-mediated EPSP prevented a spike from occurring, even
when a sizeable NMDA-mediated EPSP remained (Fig. 6A, same
cell as in Fig. 4, E and F). Figure 6B
shows a MGD stellate neuron where DNQX largely abolished the EPSP.
Conversely, in a different neuron, blocking NMDA receptors with APV
slightly reduced the EPSP amplitude but significantly reduced the
EPSP's area and duration (Fig. 6D, middle vs.
bottom). The effect of APV persisted even at hyperpolarized
membrane potentials for suprathreshold and subthreshold IC inputs. From
the same neuron as in Fig. 6D, Fig. 6E
demonstrates that blocking the NMDA component effectively prevented the
Ca2+ burst at
86 mV. An IC-evoked NMDA component at
hyperpolarized membrane potentials was seen in three other cells tested
in APV.
We also tested the effect of the monosynaptic GABAA IPSP
(Peruzzi et al. 1997) on the IC-evoked EPSP. Bath
application of the GABAA receptor antagonists bicuculline
or SR95531 resulted in a larger, longer lasting EPSP in cells with
IN/EX IC inputs (Fig. 6, D and E, middle,
and C, 5/5 cells tested) and with EX/IN IC inputs (not
shown, 2/2 cells tested), but they had little effect on cells with EX/O
IC inputs (2/3 cells tested). In two MGD neurons, blocking
GABAA receptors uncovered a suprathreshold EPSP (Fig. 6,
D and E). In the MGV, however, the EPSPs revealed
by drug application were subthreshold at rest (Fig. 6C,
n = 5).
Timing of BIC evoked first-spike latencies
Typically, because of the Ca2+ burst and other
voltage-dependent conductances, synaptically generated first-spike
latencies of thalamic neuron are highly dependent on membrane potential (Fig. 7, A-C) (Hu et
al. 1994; Jahnsen and Llinas 1984a
,b
;
McCormick 1991
). However, two neurons with IN/EX IC
input exhibited exceptionally consistent first-spike latencies that
were independent of the membrane potential (Fig. 7, D-F).
Figure 7A illustrates a MGD tufted cell with an EX/O IC
input. Cells with this input were typically suprathreshold at both
depolarized and hyperpolarized membrane potentials. The first-spike
latency (FSL) was fairly consistent at membrane potentials depolarized
above levels where the Ca2+ burst would be strongly
activated (Fig. 7C, points above
70 mV). When
hyperpolarized into the calcium burst mode of firing, cells increased
their FSL as hyperpolarization increased (Fig. 7C, points
below
70 mV). Changes in FSL >10 ms were observed in some cases. The
increased FSL appeared to be caused by slow activation of the
Ca2+ burst (Fig. 7B). In contrast to the EX/O
cells, IN/EX and EX/IN cells usually generated synaptically induced
spikes only at membrane potentials positive to
60 mV. Figure 7,
D-F, illustrates one of the two neurons with IN/EX IC input
that was suprathreshold at rest and its unusual spike response. Over a
40 mV range, the FSL change was only 400 µs. Even when in the
Ca2+ burst firing mode (Fig. 7E), the action
potential was evoked at a fixed latency. Only the first spikes were
well timed, later spikes were not (Fig. 7D, later spikes).
|
Although we did not investigate the effects of bicuculline on the neurons with well-timed FSLs, we did look at FSLs of one neuron with IN/EX IC input and one with EX/IN IC input during blockade of GABAA. In these two neurons, one of which is shown in Fig. 6, D and E, middle, the timing of FSL was similar to that of the EX/O neurons. This result supports the idea that the inhibition is necessary to produce the consistent first spike latency.
Descending inputs to MGB neurons: corticothalamic EPSPs and TRN IPSPs
In horizontal or sagittal slices, we also could stimulate the descending corticothalamic (CT) and thalamic reticular nucleus (TRN) inputs in the thalamic radiations. Activating these descending axons elicited a much more standardized response than IC stimulation, eliciting a GABAA IPSP/EPSP/GABAB IPSP sequence in 74% of both tufted and stellate neurons throughout the MGB (Fig. 8, A and B, and Table 3). The remainder received only excitatory corticothalamic input (EX/O, 20%) or only inhibitory TRN input (IN/O, 5%). Again, we cannot be certain whether EX/O and IN/O patterns reflect the true innervation or the selective stimulation of one fiber type.
|
Postsynaptic potential latencies were fairly uniform, regardless of neuron location or anatomic type (Table 3). Unlike IC inputs, there was no significant difference in the corticothalamic EPSP latency for neurons with IN/EX and EX/O patterns. Nor was there a significant difference in the TRN IPSP latency between any group. Inhibitory TRN inputs consistently had significantly shorter latencies than corticothalamic excitatory inputs (Fig. 8, A and B, Table 3), indicating that TRN axons conduct faster than corticothalamic axons.
As shown in Fig. 8, C and D, stimulation of corticothalamic axons generated a graded response, with significantly more discernible levels of excitation (5.8 ± 1.7) than excitatory IC inputs (P < 0.0001, Mann-Whitney test). Likewise, blocking glutamatergic transmission using channel blockers APV and DNQX to isolate the GABAergic TRN input revealed multiple amplitude levels for GABAA and GABAB IPSPs (4.0 ± 1.4, n = 5). The number of amplitude levels for TRN inputs was statistically similar to the number observed for IC IN/O responses. The increases in GABAA and GABAB IPSPs paralleled each other, suggesting that single terminals activates both types of GABA receptor postsynaptically. Isolation of TRN inputs with APV and DNQX also illustrated that, like IC inhibitory inputs, descending TRN inputs are monosynaptic to relay neurons (Fig. 8, E and F).
Pharmacology of descending inputs
Bath application of receptor antagonists confirmed that the
short-latency IPSP activated GABAA receptors
(n = 9, Fig. 10) and the long-latency IPSP activated
GABAB receptors (n = 6, not shown). Like
the IC excitatory input, corticothalamic inputs to both types of MGB
neurons had distinguishable AMPA and NMDA components with considerable
variation in the contribution of each component for individual neurons.
NMDA receptor antagonists consistently caused a decrease in the
duration of the corticothalamic-evoked EPSP in both MGV (Fig.
9, A and B) and MGD
neurons (Fig. 9C) and occasionally decreased the peak
amplitude. (Fig. 9B). Coapplication of APV and DNQX
eliminated the EPSP completely (n = 5, Fig.
8E). Blocking NMDA receptors produced effects even at
membrane potentials (less than 70 mV) where NMDA receptors in other
brain regions are blocked by Mg2+ ions (Fig. 9,
bottom). As illustrated in Fig. 9B, addition of APV prevented a Ca2+ burst activation at
77 mV. Thus the
NMDA components of both IC and corticothalamic inputs have an unusual
voltage dependence. Furthermore the NMDA component can contribute to
the spike response of the burst depolarization.
|
Timing of suprathreshold corticothalamic responses
Given the modulatory function typically attributed to
corticothalamic inputs, it was surprising that suprathreshold responses were evoked in a number of MGB cells by corticothalamic axon
stimulation (Figs. 9C and
10). Like the suprathreshold responses
to the EX/O IC inputs (Fig. 7, A-C) and unlike the
suprathreshold responses to IN/EX IC inputs (Fig. 7, D-F),
the latency of the corticothalamic-evoked first spike was dependent on
membrane potential, regardless of the presence or absence of a
preceding GABAergic event (Fig. 10). Cells of either morphology in both
dorsal and ventral divisions could be suprathreshold in response to
stimulation of the descending inputs. Figure 10A illustrates
synaptic and suprathreshold responses to shock stimulation of the
descending input as the cell was moved around rest in normal saline and
while the GABAA IPSP was blocked. In normal saline, at
potentials more depolarized than 70 mV, the FSL was fairly
consistent, with a typical jitter of 1-2 ms (Fig. 10B).
Like the spikes generated by excitatory IC inputs in EX/O cells, the
FSL of CT-evoked spikes increased as the cell was hyperpolarized due to
slow activation of the regenerative Ca2+ depolarization.
The latency increase and the jitter when hyperpolarized were not
significantly affected by the TRN GABAA IPSP (Fig.
10B) because they were unchanged in bicuculline. The
inhibitory TRN input did, however, make the production of synaptically
evoked spikes less likely at membrane potentials between
55 and
70 mV (Fig. 10).
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DISCUSSION |
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Analysis of MGB morphology
Intracellularly recorded and filled MGV and MGD neurons did not
differ in soma size or shape, with pyramidal and round somata of
various sizes in both divisions. Similar results were reported recently
(Clerici and Coleman 1998) for Nissl-stained rat
neurons. In another study, MGD neurons were slightly but significantly smaller than MGV neurons (Senatorov and Hu 1997
).
Because our results are based on a relatively small number of cells
throughout the dorsal and ventral divisions, we may not have been able
to detect small divisional differences. However, we reiterate that there were large individual variations in neuron areas without any
consistent divisional differences in soma size or shape.
Quantitative analysis of dendritic morphology provided support for
described qualitative differences in MGB cell morphology (Clerici et al. 1990; Morest 1984
), and a
way to distinguish tufted from stellate neurons. Such a quantitative
analysis allowed a more objective correlation of morphology with
physiology and location for individual cells. Tufted neurons in the MGV
and MGD had shorter dendrites, a smaller dendritic tree area, more
profuse branching, and greater dendritic orientation compared with
stellate neurons, which were only found in MGD. In contrast to other
anatomic studies (Clerici and Coleman 1990
; Hu et
al. 1994
; Morest 1964
, Morest 1965a
;
Winer and Morest 1983
) most of our labeled neurons were recorded and drawn in the horizontal plane because we wanted to drive
synaptic inputs. However, we recorded from and drew a small number of
neurons from coronal and sagittal slices and observed no differences in
quantitative dendritic characteristics. Although tufted neuron
dendrites were significantly more polarized than stellate dendrites,
they were not as strongly polarized as Golgi studies of rat and cat
have suggested. A lack of polarization was most evident at <50 µm
from the soma, where tufted neuron dendrites sometimes projected in all
directions. One reason that we observed longer dendrites and less
oriented dendritic trees may be that we analyzed single cells from
relatively thick sections (400-500 µm). A previous rat Golgi study
used 100- to 150-µm sections (Clerici et al. 1990
) and
labeling of many neurons in each section restricts analysis of
dendrites to single sections. Such restrictions could omit significant
portions of the dendritic tree found in adjacent sections. Anatomical
studies support the existence of laminae in MGV (Clerici and
Coleman 1990
; Morest 1965a
), which are thought
to be the anatomic substrate of isofrequency laminae recorded in vivo
(Aitkin and Webster 1971
, 1972
). Although the MGV may
have cell laminae and numerous neurons with dendrites projecting
parallel to those laminae, the overlap of inputs received by neurons in
adjacent laminae is probably substantial on dendrites that project
orthogonally to the laminae. In the rat, dendritic tree orientation may
not be as significant a factor in contributing to the narrow frequency
response as long as the proximal dendrites lie within a single cell lamina.
Differences in the dendritic characteristics of tufted and stellate
neurons resemble some of those described for cells in visual and
somatosensory thalamus. Somatosensory neurons in the rat medial
division of the posterior thalamic group and ventrobasal complex had
different morphologies but responded similarly to tactile input
(OHara and Havton 1994). In contrast, cat dorsal LGN (dLGN) neurons have anatomic features that are correlated fairly
well with their visual responses (Friedlander et al.
1981
; Humphrey and Weller 1988
),
including specialized dendritic appendages that are involved in triadic
synapses with interneurons. Rat MGB neurons lack such specialized
dendritic appendages and are more similar to rat somatosensory neurons.
The morphological differences between tufted and stellate MGB cells may
reflect differences in how afferents converge on them, but it is hard
to know whether this would cause PSPs to integrate differently to
affect acoustic response properties.
Intrinsic properties
Overall, the membrane characteristics we observed were similar to
those reported for a handful of neurons in medial geniculate and a very
large number of neurons in other thalamic regions (Crunelli et
al. 1987; Jahnsen and Llinas 1984a
,b
;
Pape and McCormick 1995
; Turner et al.
1997
). However, in contrast to the previous results described
from the MGB (Hu 1995
; Hu et al. 1994
;
Senatorov and Hu 1997
), we found that identified neurons
in the ventral and dorsal divisions of the MGB have similar intrinsic
properties, with only a few consistent differences (Table 2).
Furthermore these properties were not segregated by cell morphology.
The main difference in intrinsic properties was an increased prevalence of a sag potential in MGV cells. This sag was the only intrinsic property correlated with morphology. Only tufted neurons, which included MGV and some MGD cells, displayed a sag, but not all tufted
neurons displayed a sag.
A discussion of the possible discrepancies between our results and
those of the only other studies comparing intracellular characteristics
of MGV and MGD neurons is warranted because those studies have implied
that differences in intrinsic features could account for the nature of
the different responses to auditory stimuli seen in vivo when recording
from dorsal versus ventral division cells (Hu 1995;
Hu et al. 1994
). One possible explanation for some of
the differences is the [K+] of the salines, which has a
controlling influence on resting membrane potential. However, changing
our [K+] to match that of the previous studies did not
create populations of MGB neurons that were resolvable by resting
potential, firing mode, or subdivision nor did it alter our finding
that both MGV tufted and MGD stellate and tufted neurons could be in
burst or tonic mode.
A second possible explanation for the differences could be the type of
preparation used. Our recordings were done in a 400- to 500-µm slice
taken in any of the three standard planes and from cells in all regions
of the dorsal and ventral divisions. Hu and colleagues used an
"explant" preparation (Hu 1995; Hu et al.
1994
), where a portion of the unilateral diencephalon
containing the entire brachium of the IC and medial geniculate was
removed and isolated. With this preparation, they only recorded from
the caudal two-thirds to three-quarters of the MGB because rostral MGB
is obscured by the lateral geniculate nucleus and the brachium of the
superior colliculus (Paxinos and Watson 1986
) and only sampled cells
400-500 µM deep to the free surface of the MGB. Thus
access was restricted to the caudodorsal MGD and the caudal portion of
the lateroventral division of the MGV (Clerici and Coleman 1990
). Such an explant preparation would leave more MGB circuitry intact, but it is not clear whether structures deeper than
400-500 µM would still be viable. When we restricted our analysis to
those neurons that are accessible in the explant preparation, we still
found that MGV and MGD neurons had similar membrane characteristics except for the increased percentage of MGV neurons expressing a sag.
Therefore differences in the regions sampled do not appear to cause the
conflicting results. In addition, differences in morphology were not
correlated with differences in membrane properties because a comparison
of identified MGV tufted and MGD stellate neurons yielded identical
results to the full populations of MGV and MGD neurons.
Another possible explanation is in the choice of experimental data. By
comparing a selected subset of MGV cells that were in single spike mode
and MGD cells that were in burst mode, a mean resting membrane
potential that was 9 mV more negative in MGD cells was noted (Hu
1995). We also note a similar difference in membrane potential
when comparing bursting versus tonic firing neurons (see
RESULTS). However, our data taken from all sampled cells do
not show such a difference in membrane potential between areas or
between cell types. Because 20-30% of MGV neurons in the explant
preparation were in the burst firing mode (Hu et al. 1994
), similar to the proportion found in our study (Table 2), exclusion of MGV bursting neurons when comparing MGV and MGD resting potentials may have made the populations appear more segregated than
the total population of MGV and MGD neurons. Using sharp microelectrodes, the difference in potentials was attributed
to the presence of a Cs+-sensitive sag potential
(Ih) found only in MGV neurons that was active
at resting potential (Hu 1995
). We also have found the sag mainly in MGV neurons, but our data indicate that tufted cells in
MGD also can display a sag potential. A whole cell recording study also
found enhanced activity of Na+-K+-ATPase in MGD
neurons relative to MGV neurons (Senatorov and Hu 1997
).
However, using sharp microelectrodes, the small divisional difference
in Na+-K+-ATPase currents would not contribute
to a significant difference in resting potentials. Regardless of cell
morphology or location in the MGB, synaptic stimulation of neurons in
the present study could evoke bursts at hyperpolarized membrane
potentials and one or more Na+ spikes at depolarized
potentials. Together these data would bring into question the notion
that membrane potential, controlled by intrinsic features of cells in
dorsal versus ventral MGB, is a controlling influence in response
differences seen in vivo.
Thus in vivo, synaptic control of resting membrane potential is
probably a more powerful determinant of neuronal response than
intrinsic properties. Rat thalamic neurons receive cholinergic inputs
from the laterodorsal tegmentum and pedunculopontine nucleus and
noradrenergic inputs from the locus coeruleus (McCormick
1992). Both of these modulatory pathways would be preserved in
the explant preparation but not in the slice preparation, but the
nuclei are also deep enough that they might be adversely affected in
the explant. Mooney et al. (1995)
indicated that MGV
neurons were depolarized out of the burst range by application of
muscarinic acetylcholine receptor agonists, whereas MGD neurons were
hyperpolarized. In addition, activation of
-adrenoreceptors in
thalamocortical neurons can cause depolarization, whereas
-adrenoreceptors also can cause depolarization by a positive shift
in the activation curve of Ih (McCormick
1992
). Because the difference between MGD and MGV neuron
resting potentials in the explant preparation was due to the strong
activation of Ih in MGV tufted neurons, it is possible that selective activation of
-adrenoreceptors was able to
cause the observed differences in resting potential. Until an in vivo
MGB intracellular recording study is done, we cannot know whether MGB
neurons normally are maintained at different membrane potentials. Such
a study also would help to determine the cause(s) of the increased
latency of MGD neuron responses to sound, such as a long-latency
Ca2+ burst (Hu 1995
; Hu et al.
1994
) or the use of suboptimal acoustic stimuli to activate MGD neurons.
Inputs from the IC
BIC stimulation elicited excitatory and inhibitory synaptic events
in MGV and MGD neurons, and we have shown previously (Peruzzi et
al. 1997) that they arise from monosynaptic IC inputs. Here we
analyze the relationship of the inhibitory and excitatory IC projections. BIC stimulation most commonly elicited IN/EX or EX/O responses, but EX/IN and IN/O responses also were evoked. Both MGV and
MGD displayed similar proportions of response combinations, but
throughout the MGB, suprathreshold EX/O responses were only observed in
neurons with tufted morphology. Increased synaptic amplitudes with
increased shock intensity to BIC fibers implies that IC excitatory and
inhibitory PSPs often result from convergence of a small number of
inputs. Analysis of PSP latencies implied that there are excitatory and
inhibitory IC axons that conduct at different rates. Pharmacological
blockade of GABAA IPSPs did not change the EPSP latency for
IN/EX or EX/IN inputs, implying that the excitatory fibers generating
EX/O patterns may be a different population than those producing IN/EX
patterns. Although this analysis cannot predict the interaction of
inputs that produces variable response latencies to sound observed in
vivo, it can provide some insight into the response combinations and
relative timings of inputs necessary to produce the in vivo responses. The latency of IN/EX EPSPs was significantly longer than the latency of
EX/O EPSPs, and the latency of IN/EX, IN/O, and MGV EX/IN
GABAA IPSPs was significantly shorter than the latency of
MGD EX/IN IPSPs. Anatomic evidence from cat (St. Marie et al.
1997
) has shown that a variety of sizes of both GABAergic and
non-GABAergic (presumably glutamatergic) fibers run in the BIC that are
thought to arise primarily from IC cells. Only a small percentage of
medium and small IC axons were GABAergic, but about half of the large diameter IC axons were. Thus it is probably not surprising that different EPSP and IPSP latencies are observed and that the IPSPs often
preceded EPSPs. It is also probably not surprising, given the fairly
large population of small non-GABAergic axons reported that many EPSPs
are small and subthreshold. Taken together, these results support the
notion that IC inhibition can be fast and may precede excitation under
some conditions in vivo. Certain neurons in the awake mustached bat IC
display significant facilitation when a CF pulse and its
doppler-shifted echo are presented together compared with either
stimulus presented alone (Suga et al. 1997
). This effect
was enhanced by bicuculline iontophoresis, largely due to an increase
in an APV-sensitive late component (Suga et al. 1997
).
Blockade of GABAA inhibition in our slices could uncover a
robust suprathreshold IC input with a significant APV sensitive late
component (Fig. 6, D and E).
Pharmacological analysis showed that excitatory IC inputs activated
AMPA and NMDA glutamate receptors and inhibitory IC inputs activated
GABAA and GABAB receptors. The NMDA component
was active even at hyperpolarized membrane potentials and could affect
the MGB cell's ability to respond with a Ca2+ burst (Fig.
6E). Many of the IC EPSP properties described here have been
observed in the MGB and other thalamic regions. Previous studies of rat
MGB, LGN, and ventrobasal complex (VB) neurons showed that stimulation
of ascending inputs to these regions evokes EPSPs with AMPA and NMDA
components (Hu et al. 1994; Kao and Coulter 1997
; Turner et al. 1994
). In the MGB, it was
reported that IC EPSPs in MGV neurons were largely due to AMPA receptor
activation, whereas those in MGD had a more equal mixture of AMPA and
NMDA components (Hu et al. 1994
). Although we did not
investigate this issue in detail, our results indicate that there are
clear exceptions (Fig. 6A) and that the proportion of each
component may vary between neurons. Further study is required to
determine the extent of this variability and whether the proportion of
AMPA and NMDA components is related to the MGB response pattern to IC
input observed in the slice.
Like the present study, studies of the rat and cat dorsal LGN showed a
noticeable NMDA component, even at hyperpolarized membrane potentials
<75 mV, that was occasionally necessary to evoke a Ca2+
burst in response to optic tract stimulation (Esguerra et al. 1992
; Turner et al. 1994
). In these LGN neurons,
the NMDA component expanded the voltage range of the burst. NMDA
antagonists applied to the cat LGN in vivo could reduce the visual
responses of X and Y neurons (Hartveit and Heggelund
1990
; Heggelund and Hartveit 1990
;
Kwon et al. 1992
). The functional role of the IC NMDA
component in the MGB is unclear because the effects of NMDA receptor
agonists or antagonists on MGB neurons in vivo have not been
investigated in detail (Suga et al. 1997
).
In agreement with a previous MGB study (Hu 1995) we
found that, for most cells with suprathreshold IC input, the latency of the BIC-evoked first spike was dependent on membrane potential and EPSP
height. In our study, suprathreshold burst responses at hyperpolarized
membrane potentials were observed mainly in neurons not receiving
inhibitory IC input (EX/O input). When depolarized, evoked action
potentials were short latency and consistent, but hyperpolarization
below
70 mV greatly increased the FSL over a small voltage range
probably because of activation of the regenerative Ca2+
burst and the increased time for the burst to reach spike threshold. If
neurons with EX/O inputs actually receive little or no IC inhibition in
vivo, then a burst response may be useful for the amplification and
detection of weak stimuli, which would not require temporal precision.
Receiver operating characteristics analysis of cat LGN cells has shown
that the burst mode is suited for signal detection (Guido et al.
1995
) and that LGN neurons can burst during waking, especially
during the initial fixation to a visual stimulus (Guido and
Weyand 1995
). The nonlinear increase in FSL with
hyperpolarization and its interplay with EPSP height makes it unlikely
that the burst response is an analogue to temporal converter (Hu
1995
) because multiple combinations of EPSP height and membrane
potential could produce the same FSL.
We noted that some neurons with inhibitory IC inputs could fire
short-latency action potentials at more depolarized membrane potentials, usually greater than 55 mV, and in two cases, the neurons
responded over a wide range of membrane potentials both above and below
rest. For these two neurons, the FSL was independent of membrane
potential. The unusual short-latency response at hyperpolarized potentials appeared to be caused by the combination of depolarizing inhibition rapidly activating the Ca2+ burst followed
closely by a large excitatory input. Moreover, responses at different
membrane potentials may converge to produce a more uniform starting
point on which the EPSP is superimposed. Further experiments are
necessary to determine whether blocking inhibition will abolish the
fixed FSL and whether the IPSP and EPSP latencies affects the timing
consistency. However, because block of GABAA receptors in
two neurons with IC inhibition revealed poorly timed suprathreshold
responses (Fig. 6, D and E), the early IPSP
probably has a significant role in generating well-timed first spikes.
In the LGN, consistently timed spikes are associated with the tonic
firing mode, a state believed to be suited for signal analysis
(Guido et al. 1995
). Because these IN/EX cells seem to
be combining signal amplification and detection capabilities provided
by the burst mode with the consistently timed spikes useful for signal
analysis, it implies that they may be able to consistently analyze weak
acoustic stimuli.
Inputs from the cortex and the thalamic reticular nucleus
Stimulation of the thalamic radiations excited corticothalamic and
TRN fibers, causing an IN/EX response in most neurons. Overall, the
corticothalamic excitatory input had more distinguishable amplitude
levels than IC input as the stimulus intensity increased, implying that
more corticothalamic fibers converge onto single MGB neurons. Like the
ascending IC input, the excitatory corticothalamic inputs activated
AMPA and NMDA receptors, whereas the inhibitory TRN inputs activated
GABAA and GABAB receptors. The NMDA component of the corticothalamic excitatory input also had an unusual voltage dependence with a detectable APV-sensitive component even at membrane potentials below 70 mV. Like the EX/O IC input, the FSL of
corticothalamic action potentials was sensitive to membrane potential,
regardless of whether TRN inhibition was present.
Although this is the first intracellular description of descending
input to MGB neurons, studies in other thalamic regions have
investigated the corticothalamic and TRN inputs. Stimulation of the
internal capsule or cortex causes a combination of EPSPs and IPSPs in
somatosensory, motor, and anterior thalamic regions (Deschenes
and Hu 1990; Kao and Coulter 1997
; Pare
et al. 1991
; Warren et al. 1994
). In these
studies, the EPSP usually preceded the IPSP. We showed that the IPSP
consistently preceded the EPSP. This discrepancy is probably due to the
different placement of the stimulating electrodes. Placement in the
cortex or internal capsules would necessitate synaptic activation of
TRN neurons that then would project to thalamic neurons. Our placement
in the thalamic radiations directly activated TRN axons. Our responses suggest that TRN axons conduct faster than most corticothalamic axons.
Isolated corticothalamic inputs in other thalamic regions were shown to
be composed of many smaller events (Deschenes and Hu
1990
; Warren et al. 1994
), similar to our
results for MGB neurons.
Stimulation of the descending inputs in other thalamic areas also has
been shown to activate AMPA and NMDA receptors via cortical input and
GABAA and GABAB receptors via TRN input.
GABAA receptor blockade or destruction of the TRN resulted
in substantial increases in EPSP amplitude that could uncover a
suprathreshold response (Deschenes and Hu 1990;
Eaton and Salt 1996
; Warren et al. 1994
), an effect that we also have observed (Fig. 10A). In these
studies, the NMDA contribution to the corticothalamic EPSP ranged from moderate (Deschenes and Hu 1990
; Eaton and Salt
1996
), which was observed most often in the present study (Fig.
9), to dominant (Kao and Coulter 1997
). Voltage-clamp
analysis of cortical inputs to rat VB neurons revealed substantial NMDA
currents between
50 and
80 (Kao and Coulter 1997
).
One possible explanation is that NR2C and NR2D NMDA receptor subunits
are resistant to Mg2+ block at physiological resting
potentials and are present in the thalamus of young and adult rats
(Kao and Coulter 1997
; Monyer et al.
1994
; Wenzel et al. 1995
, 1997
). It is notable
that both the IC and cortical inputs appear to have similar unusual
NMDA channel voltage dependence, suggesting that both inputs may
activate postsynaptic NMDA receptors with similar subunit compositions.
Electrical stimulation of corticothalamic inputs can elicit
suprathreshold responses in vitro and in vivo (Deschenes and Hu 1990; Kao and Coulter 1997
; this study),
implying that these inputs could drive sensory responses of
thalamocortical neurons. This suggestion opposes the simply modulatory
role normally attributed to the corticothalamic input's function
(Crick and Koch 1998
; McCormick and Bal
1994
). Although electrical stimulation produces artificially
synchronous activation of corticothalamic axons, cortical inputs appear
to generate suprathreshold events in ventrolateral nucleus neurons
during cortical "desynchronization" in anesthetized cats
(Steriade 1997
). During desynchronization, the cortical
excitation of TRN neurons appeared to be generally weak
(Steriade 1997
), unlike the strong corticoreticular
input during sleep spindles and delta rhythms (Contreras and
Steriade 1996
; Contreras et al. 1996
;
Destexhe et al. 1998
; Steriade 1997
). A
class of possibly interconnected corticothalamic neurons has been
identified in the cat cortex that fires rhythmic high-frequency bursts
at 20-50 Hz (Steriade et al. 1998
), which might be able
to quickly synchronize corticothalamic neurons and provide a strong
excitatory corticothalamic input. Another characteristic of inputs
considered to be drivers is that they determine the receptive field of
the postsynaptic cell. In the mustached bat MGB, focal inactivation of
auditory cortex produced a best frequency (BF) shift for cells the BF
of which was slightly different from the inactivated region of cortex (Zhang et al. 1997
). Finally, cortex-dependent
input can generate activity in some cat MGB cells for hundreds of
milliseconds after the offset of acoustic stimulation, suggesting that
cortical input may cause action potentials in MGB neurons in the
absence of IC activity (Ryugo and Weinberger 1976
).
The inhibitory TRN input was present in most MGB neuron responses, and
multiple fibers appeared to converge onto a single MGB neuron (Fig. 8,
E and F). Pharmacological blockade of TRN IPSPs
invariably increased the corticothalamic EPSP and could decrease the
stimulus intensity necessary to evoke a spike or increase the ability
to spike over a greater voltage range (Fig. 9A). Although
the FSL increased as membrane potential decreased, which also occurred
for EX/O IC inputs, TRN inhibition had little effect on the
corticothalamic evoked FSL (Fig. 10). Thus TRN inhibition may not be as
strongly coupled to corticothalamic excitation as IC inhibition is to
IC excitation. It is also possible that TRN inhibition could be
associated more closely with ascending IC excitation because TRN
activation preceding click stimuli could suppress the click response in
rat MGB neurons for 20-30 ms (Shosaku and Sumitomo
1983).
Correlation of MGB anatomy and physiology
In support of the hypothesis that the dendritic architecture of
MGB neurons does not predict their in vivo responses, we found only a
few consistent differences between tufted and stellate neurons
physiologically in vitro. Only tufted neurons displayed a depolarizing
sag, as reported in previous studies (Hu 1995). Because
we found that the resting potential of tufted and stellate neurons was
similar, the significance of the sag in tufted neurons is unclear.
Perhaps the resting potential of tufted neurons can be modulated by
changing the voltage dependence of the sag activation by modulatory
neurotransmitters (Banks et al. 1993
; Pape and
McCormick 1989
), the sag curtails the inhibitory duration of
GABAB inhibition, or it promotes a more robust rebound
burst after hyperpolarization (Luthi and McCormick
1998
). The latter hypothesis is less likely because we
did not observe an enhanced burst frequency or number of spikes in MGV
neurons compared with MGD neurons.
Only a subset of tufted neurons were capable of responding to BIC
stimulation with a large all-or-none EPSP that was often suprathreshold. These EPSPs may be the physiological equivalent of
large excitatory afferents observed at the light and electron microscopic levels in the cat and hamster (Campbell
and Frost 1988; Jones and Rockel 1971
;
Majorossy and Kiss 1976
) and at the light level in
ferret and guinea pig (Malmierca et al. 1997
;
Pallas and Sur 1994
). It is possible that MGD tufted
neurons receiving this large excitatory input are those that project to
layer IV of primary auditory cortex (Mitani et al.
1987
). Such a projection would blur the classification of MGV
as a lemniscal or first-order thalamic nucleus and MGD as an
extralemniscal or higher-order thalamic nucleus (Guillery
1995
). Finally, one of the neurons with a fixed FSL at all
potentials had stellate morphology and was found in the MGD. The
general lack of correlation of morphology or MGB subdivision with
intrinsic physiology implies that most MGB neurons can perform a
similar spectrum of transformations of synaptic input. It further
indicates that MGB acoustic responses are dictated mainly by the
combination and pattern of synaptic inputs that MGB neurons receive.
Future studies should investigate whether MGB neurons might receive
specific combinations of input designed to process particular stimulus
features. One might expect that neurons receiving EX/O IC inputs would
process inputs in a manner similar to LGN or VB neurons receiving EX/O
retinal or trigeminal input, but MGB neurons receiving IN/EX inputs
might have more complex responses reflecting their unique pattern of innervation from ascending inputs.
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ACKNOWLEDGMENTS |
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We are indebted to J. Ekleberry, J. Meister, and I. Siggelkow for excellent technical assistance. We thank D. Uhlrich and R. Guillery for helpful comments on a draft of the manuscript and W. Rhode for the use of his Neurolucida system.
This work was supported by the National Institute on Deafness and Other Communications Disorders Grant DC-00116 and funds provided by a grant to the University of Wisconsin Medical School from the Howard Hughes Medical Institute Research Resources Program for Medical Schools.
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FOOTNOTES |
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Address for reprint requests: P. H. Smith, Dept. of Anatomy, University of Wisconsin-Madison, Madison, WI 53706-1532.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 August 1998; accepted in final form 13 January 1999.
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REFERENCES |
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