Department of Cell Biology and Anatomy and Neuroscience Center of Excellence, Louisiana State University Medical Center, New Orleans, Louisiana 70112
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ABSTRACT |
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Lo, Fu-Sun, William Guido, and Reha S. Erzurumlu. Electrophysiological Properties and Synaptic Responses of Cells in the Trigeminal Principal Sensory Nucleus of Postnatal Rats. J. Neurophysiol. 82: 2765-2775, 1999. In the rodent brain stem trigeminal complex, select sets of neurons form modular arrays or "barrelettes," that replicate the patterned distribution of whiskers and sinus hairs on the ipsilateral snout. These cells detect the patterned input from the trigeminal axons that innervate the whiskers and sinus hairs. Other brain stem trigeminal cells, interbarrelette neurons, do not form patterns and respond to multiple whiskers. We examined the membrane properties and synaptic responses of morphologically identified barrelette and interbarrelette neurons in the principal sensory nucleus (PrV) of the trigeminal nerve in early postnatal rats shortly after whisker-related patterns are established. Barrelette cell dendritic trees are confined to a single barrelette, whereas the dendrites of interbarrelette cells span wider territories. These two cell types are distinct from smaller GABAergic interneurons. Barrelette cells can be distinguished by a prominent transient A-type K+ current (IA) and higher input resistance. On the other hand, interbarrelette cells display a prominent low-threshold T-type Ca2+ current (IT) and lower input resistance. Both classes of neurons respond differently to electrical stimulation of the trigeminal tract. Barrelette cells show either a monosynaptic excitatory postsynaptic potential (EPSP) followed by a large disynaptic inhibitory postsynaptic potential (IPSP) or just simply a disynaptic IPSP. Increasing stimulus intensity produces little change in EPSP amplitude but leads to a stepwise increase in IPSP amplitude, suggesting that barrelette cells receive more inhibitory input than excitatory input. This pattern of excitation and inhibition indicates that barrelette cells receive both feed-forward and lateral inhibition. Interbarrelette cells show a large monosynaptic EPSP followed by a small disynaptic IPSP. Increasing stimulus intensity leads to a stepwise increase in EPSP amplitude and the appearance of polysynaptic EPSPs, suggesting that interbarrelette cells receive excitatory inputs from multiple sources. Taken together, these results indicate that barrelette and interbarrelette neurons can be identified by their morphological and functional attributes soon after whisker-related pattern formation in the PrV.
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INTRODUCTION |
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The stereotypic arrangement of whisker
follicles and sinus hairs on the snout of nocturnal rodents is
replicated by the distribution of presynaptic afferent arbors and their
postsynaptic target cells ("barrelettes") in the brain stem
trigeminal complex (BSTC) (Bates and Killackey 1985;
Belford and Killackey 1979
; Erzurumlu et al. 1980
; Ma 1993
; Ma and Woolsey
1984
). Trigeminothalamic projection (barrelette) cells of the
principal sensory nucleus of the trigeminal nerve (PrV) relay
whisker-specific patterns to the dorsal thalamus ("barreloids"),
and consequently to the primary somatosensory cortex ("barrels")
(Belford and Killackey 1980
; Durham and Woolsey 1984
; Erzurumlu et al. 1980
; Erzurumlu
and Jhaveri 1990
; Erzurumlu and Killackey 1980
;
Ivy and Killackey 1981
; Killackey and Fleming 1985
; Ma and Woolsey 1984
; Senft and
Woolsey 1991
; Van der Loos 1976
; Woolsey
and Van der Loos 1970
). This patterned neural organization takes place during a sensitive period in development. Disruptions of
the sensory periphery before postnatal day (PND)
3-4 irreversibly alter central neural patterns (see
Erzurumlu and Killackey 1982
; Jhaveri and
Erzurumlu 1992
; O'Leary et al. 1994
;
Woolsey 1990
for reviews).
Whisker-related primary afferents convey the pattern template to
select groups of target neurons at all levels of the trigeminal neuraxis (Erzurumlu and Jhaveri 1990). In the rat,
barrelette formation begins shortly before birth and it is consolidated
by PND5 (Belford and Killackey 1980
;
Chiaia et al. 1992
). To understand the mechanisms
underlying pattern formation in the mammalian CNS, it is important to
distinguish between the structural and functional characteristics of
pattern forming neurons and other cells. In this study we focused on
the barrelette and interbarrelette neurons in early postnatal rat PrV.
Using whole cell patch recording, immunohistochemistry, and
intracellular biocytin labeling techniques, we charted out the
morphological characteristics, membrane properties, and synaptic
circuitry within barrelette region of the PrV of PND4-12
rat pups. We show that barrelette and interbarrelette cells can be
distinguished by their morphological and electrophysiological properties shortly after whisker-related pattern formation. Our analyses of synaptic responses also suggest that barrelette cells receive excitatory input from a single whisker follicle, and a strong
lateral inhibition originating from neighboring whiskers. Interbarrelette cells receive excitatory inputs from a variety of
sources, including multiple whisker follicles, other interbarrelette or
barrelette cells. In both types of cells, the excitation is mediated by
N-methyl-D-aspartate (NMDA) and non-NMDA
receptors, whereas the inhibitory component is mediated by
GABAA receptors.
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METHODS |
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Brain slice preparation
Sprague-Dawley rat pups ranging in age from PND4 to 12 were deeply anesthetized with Fluothane (Halothane) and then killed by decapitation. The brain was removed quickly and immersed in cold (4°C), sucrose-based artificial cerebrospinal fluid (ACSF, in mM: 234 sucrose, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, 24 NaHCO3, 11 glucose, and 0.5 CaCl2) bubbled with 95% O2-5% CO2, pH 7.4. The brain stem was embedded in 2% agar and cut into 500-µm-thick transverse sections with a vibratome (Electron Microscopy Sciences). Slices containing the trigeminal principal sensory nucleus (PrV) were placed in a submerged-type recording chamber (Fine Science Tools) and continuously perfused (2 ml/min) with normal ACSF (in mM: 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 26 NaHCO3, 10 glucose, and 2 CaCl2, pH 7.4) at room temperature. Bath application of bicuculline (10 µM) or D-2-amino-5-phosphonovaleric acid (D-APV; 50 µM) were used to block GABAA or NMDA receptors. We also applied Cs+ (1 mM), Ni2+ (200-500 µM) or 4-aminopyridine (4-AP; 1 mM) to block H, T, and A-type conductances. A photomicrograph of the slice preparation placed in the recording chamber is shown in Fig. 1. At the ages tested, the trigeminal tract (TrV) and PrV barrelette region can be readily distinguished.
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Electrophysiological methods
Recordings began at least 1 h after incubation in normal
ACSF. Whole cell patch micropipettes were pulled horizontally in two
stages from borosilicate glass (WPI, K150F-4) with a P-87 puller
(Sutter Instrument). The patch electrodes were backfilled with a
potassium-based solution (in mM: 140 K-gluconate, 10 HEPES, 1.1 EGTA-Na, 0.1 CaCl2, 2 MgCl2, 2 ATP-Na, and 0.2 GTP-Na, with or without
1% biocytin, pH 7.25) with a tip resistance of 7-10 M. Neurons in
the ventral part of the PrV (barrelette region) were blindly patched
using the techniques described by Blanton et al. (1989)
and Ferster and Jagadeesh (1992)
. In brief,
patch-electrode resistance was monitored in Bridge Mode of Axoclamp 2B
amplifier by measuring the voltage drop induced by a current pulse
(
100 pA, 200 ms). An increase in resistance of 20-50 M
was taken
as a sign that the electrode tip contacted the surface of a neuron. A
steady negative pressure was applied with a 5-ml syringe to form a
gigaohm seal. Then a brief suction was used to break into the cell
body. The formation of whole cell configuration was indicated by a
sudden drop in seal resistance and a DC drop of >55 mV. After "break-in," the serial resistance was compensated with bridge balance, and junction potential (Neher 1992
) was not
corrected. We only collected data from cells with resting membrane
potential negative to
55 mV and input resistance >300 M
. Neuronal
activity was digitized with an Instrutek VR10B interface unit and
stored on a Macintosh Power PC (9500/132) using Pulse (HEKA)
software program. For biocytin labeling experiments, we filled the
patch electrodes with 1% biocytin dissolved in potassium-based
solution. Once membrane properties and synaptic responses were
characterized, the cells were filled intracellularly with biocytin by
passing AC pulses (±1 nA, 60 ms for each cycle) through the
biocytin-filled recording electrode.
A pair of fine-tip stimulating electrodes (0.5 M, WPI, IRM33A05KT)
were inserted at various points along the trigeminal tract (TrV)
lateral to the ventral PrV (barrelette region). Current pulses
(0.2-0.5 ms duration, 0.05-1.0 mA) were passed through the electrodes
at 0.33 Hz to evoke postsynaptic potentials. To investigate the voltage
dependency of the postsynaptic potentials, DC current was passed
through the recording patch electrode to change the membrane potential.
Different DC pulse protocols were used to induce active conductances of
trigeminal neurons. Each cell's membrane potential was held at
60 mV
(except where indicated) to compare voltage-dependent conductances and
postsynaptic potentials between different cells.
Identification of excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) was based on their voltage dependency and their responses to glutamate and GABA antagonists. The non-NMDA component of an EPSP increased in amplitude (see Fig. 6A) with membrane hyperpolarization, enabling us to distinguish between an EPSP and a shock artifact. EPSPs showed predictable changes in amplitude with changes in membrane potential, whereas shock artifacts did not. The NMDA component of an EPSP was identified by its nonlinear voltage dependency, slow decay time, and blockade by D-APV. The GABAA receptor-mediated IPSP was identified by its reversal with membrane hyperpolarization (~70 mV), and blockade by bicuculline. The onset of the EPSPs or IPSPs was estimated by superimposing the PSPs at different membrane potentials. Despite the changes in amplitude, the onset of PSPs remained fixed. Additionally, the onset of the IPSP could be estimated by comparing responses in the presence or absence of bicuculline (see Fig. 6B).
To investigate stimulus-response relationships, we first determined the
threshold of activation (~0.05 mA, 0.3 ms) for postsynaptic potentials. We then increased stimulus intensity progressively until
the postsynaptic potential reached its maximal amplitude (approximately
<1 mA, 0.3 ms). This procedure was repeated three to five times to
ensure that the stepwise increase in amplitude was reliable and not due
to fluctuations in EPSP amplitude (see also Allen et al.
1977; Bartlett and Smith 1999
; Mock et
al. 1997
).
Histological methods
One hour after biocytin injection, the slice was fixed by 4%
paraformaldehyde in 0.1 M phosphate buffer for 48 h. The fixed slice was transferred into phosphate-buffered saline (PBS) at 4°C and
then incubated in 10% methanol + 3%
H2O2 overnight. After several rinses in PBS, the slice was reacted with avidin-biotin complex
(ABC Elite kit, Vector Laboratories) overnight at 4°C (1:100 in PBS
with 1.8% NaCl and 0.5% Triton X-100). The next day, the slice was
rinsed again in PBS and 0.1 M acetate buffer (pH 6.0) and incubated in
glucose oxidase-nickel ammonium sulfate and diaminobenzidine until the
labeled cells could be visualized. The slice was rinsed in acetate
buffer and PBS, mounted on a slide, dehydrated, and coverslipped.
Labeled cells were drawn with a drawing tube attached to a Nikon
Labophot microscope. Counterstaining for cytochrome oxidase
histochemistry (Wong-Riley and Welt 1980) revealed the
barrelettes in the PrV of PND4-12 rat brain stem.
For GABA immunohistochemistry, PND4-7 pups (n = 6) were killed with an overdose of pentobarbital sodium and perfused transcardially with PBS and 4% paraformaldehyde in PBS. The brain stems were taken out, cryoprotected in 30% sucrose in PBS, and frozen sectioned at a thickness of 50 µm. Sections were first soaked in 4% goat serum in PBS (1 h), then in anti-GABA antibody (Eugene Tech. International, dilution 1:2,000) in 1% goat serum and PBS, overnight at room temperature. The next day, sections were rinsed in PBS and incubated in biotinylated goat-anti-guinea pig antibody in PBS (Vector Laboratories, dilution 1:200) for 2 h. After several rinses in PBS, sections were processed with an avidin-biotin complex (Vectastain kit, Vector Laboratories), and the reaction product was visualized with diaminobenzidine. Control sections were processed as described above, but without the primary antibody.
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RESULTS |
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Morphological features of barrelette, interbarrelette, and
GABAergic interneurons of the PrV are illustrated in Fig.
2. Using the blind
whole cell recording technique, we could study only the medium and
large size neurons of PrV, and not the smaller GABAergic cells.
GABAergic interneurons are present throughout the nucleus and are found
within and between barrelettes (Fig. 2A). Interbarrelette
neurons are large cells with extensive dendritic trees that span
several barrelettes and septae (Fig. 2, B and C).
In contrast, barrelette neurons are smaller cells with shorter dendritic fields confined to a single barrelette domain (Fig. 2,
B and E). These anatomic observations are
consistent with previous findings of Arends and Jacquin
(1993), who used Lucifer yellow fills to identify barrelette
and interbarrelette cells.
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Electrophysiological identification of barrelette and interbarrelette cells
We performed whole cell patch recordings from 64 neurons in the
barrelette region of the PrV. Of these, 41 had a prominent A-type
potassium conductance (IA) as well as
an H-type mixed cation conductance
(IH; Fig.
3A). We were able to fill 10 such cells with biocytin, and based on their dendritic morphology they
all were barrelette neurons. Additionally these cells all had
IA and IH. Dendritic fields of five of these
cells were reconstructed and plotted against barrelette patches
visualized with cytochrome oxidase histochemistry (Fig. 2). The cell
illustrated in Fig. 2 (cell 1) and Fig. 3A showed
a depolarizing "sag" during membrane hyperpolarization (indicated
by H in Fig. 3A). Application of Cs+
(1 mM, n = 4) blocked this inward rectification (Fig.
4B), indicating that
barrelette cells possess a hyperpolarization-activated
IH. Frequently, at the end of the
hyperpolarizing current pulses, a hyperpolarizing tail (~150 ms) was
observed (Fig. 3A, indicated by A). When the barrelette cell
was depolarized (50 mV) after hyperpolarization (
120 mV), there was
a hyperpolarizing notch before the first spike (Fig. 3B,
indicated by arrow A), which led to a substantial delay in action
potential firing. Membrane depolarization led to a train of
Na+ spikes (Fig. 3B). As can be seen,
the frequency of spikes varied systematically with the intensity of
depolarizing current pulses. Application of 4-AP (1 mM,
n = 5) blocked completely the hyperpolarizing notch
(Fig. 4C) and decreased the latency of the initial spike, suggesting that these cells possess
IA. Barrelette cells could also be
distinguished by their passive membrane properties. The resting
membrane potential of barrelette cells was
62.1 ± 0.6 mV
(mean ± SE, n = 41, Fig. 4A), and
their input resistance was 633 ± 34.7 M
(n = 41, Fig. 4A), the latter confirming the morphological observation that barrelette cells have relatively small soma.
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The remaining 23 neurons also exhibited strong
IH (Fig. 3C). However,
membrane depolarization from a hyperpolarized state evoked a triangular
depolarization (Fig. 3, C and D, indicated by T)
with a burst of Na+ spikes riding on it.
Application of Ni2+ (200-500 µM,
n = 5) blocked this response (Fig. 4D),
suggesting that it is mediated by a low-threshold T-type
Ca2+ conductance
(IT). We injected biocytin into eight
such neurons. All of these cells had large dendritic trees distributed
over and in between several barrelettes, and possessed a large
IT. Thus we classified neurons with
IT as interbarrelette cells. The resting membrane potential of interbarrelette cells was 60.4 ± 0.7 mV (n = 23), which is not significantly different
from that of barrelette cells (Fig. 4A). The input
resistance of interbarrelette cells was significantly lower (2-tailed
t-test, P < 0.0002) than barrelette cells
(417 ± 34.7 M
; n = 23, Fig. 4A),
confirming the anatomic observation that interbarrelette cells have
larger call bodies than barrelette neurons.
During postnatal development (PND4-12), the resting
membrane potential and active conductances for both classes of neurons remained largely unchanged. However, we did note that input resistance declined significantly (data not shown). Ongoing studies are aimed to
detail developmental changes in electrophysiological properties of PrV
cells before, during, and after whisker-specific pattern formation
(Erzurumlu and Lo 1999).
Synaptic responses of barrelette cells
Figure 5A shows the
arrangement of stimulating and recording sites in the brain slice (see
also Fig. 1). Previous anatomic studies showed that the rat trigeminal
tract fibers are topographically organized from early embryonic ages on
(Bates and Killackey 1985; Erzurumlu and Jhaveri
1992
; Erzurumlu and Killackey 1982
,
1983
). This topographic organization has been
demonstrated by either lesions of specific whisker rows in perinatal
rats or by tracings with multiple lipophilic carbocyanine dyes placed
along the dorsoventral axis of the snout. Briefly, trigeminal fibers
carrying information from dorsal whisker rows are situated ventrally in
the tract, and those carrying information from ventral whisker rows are
located dorsally in the tract (Bates and Killackey 1985
;
Erzurumlu and Jhaveri 1992
). There is also evidence
suggesting that the rostrocaudal axis of the whisker pad is represented
along the mediolateral axis of the TrV and PrV (Bates and
Killackey 1985
; Belford and Killackey 1980
;
Erzurumlu and Killackey 1983
).
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For all barrelette cells, stimulation of the TrV evoked an EPSP followed by a long-lasting IPSP (Fig. 5B, n = 26). Occasionally, the IPSP was followed by a rebound Na+ spike (Fig. 5C). We saw no evidence of rebound low-threshold spiking confirming that barrelette cells lack IT. The IPSP reversed in polarity during membrane hyperpolarization (top traces vs. bottom traces in Fig. 5, B and C). Interestingly, the evoked responses depended on the location of the stimulation site. We marked the stimulating site near the recorded cell as site 2 and the site far from the recorded cell as site 1 (Fig. 5A). Stimulation of site 1 evoked a single IPSP (Fig. 5D), and stimulation of site 2, at the same intensity, elicited an EPSP-IPSP sequence (Fig. 5E).
We also analyzed the pharmacology of these postsynaptic potentials. In barrelette cells, stimulation of site 1 evoked an IPSP alone (Fig. 5G, trace 1) that was completely blocked by application of bicuculline (10 µM, Fig. 5G, trace 2). Thus the IPSP is exclusively mediated by GABAA receptors. Stimulation of site 2 induced an EPSP-IPSP sequence (Fig. 5H). Blocking GABAA receptors by bicuculline (10 µM) revealed a long-lasting EPSP (Fig. 5H, trace 1 vs. 2, n = 15). The late component of the EPSP was blocked by D-APV (50 µM, Fig. 5I, trace 1 vs. 2, n = 4). Therefore the EPSP is mediated by both NMDA and nonNMDA type of glutamate receptors. The evoked responses also depended on the stimulus intensity. As shown in Fig. 5, J-L, stimulation of site 1 with lower intensity induced a pure IPSP (Fig. 5J), whereas stronger stimulus evoked an EPSP-IPSP sequence (Fig. 5, J and K), the latter was probably caused by current spread in the TrV. These results indicate that the excitatory and inhibitory responses originate from different trigeminal inputs. This interpretation is supported by the following result. A progressive increase of stimulus intensity produced a stepwise increase in IPSP amplitude (Fig. 5, F and L). The number of steps suggests that a single barrelette cell receives inhibitory inputs that result from activation of at least five to seven trigeminal nerve fibers. In contrast, increasing stimulus intensity produced either little or no change (Fig. 5F) or an increase at two to three steps in EPSP amplitude (Fig. 5L, inset). This implies that barrelette cells receive inhibition originating from more trigeminal tract fibers than those for excitation.
In response to different stimulus intensities, the latency of the EPSP was nearly constant, suggesting that barrelette cells receive monosynaptic excitation from the trigeminal nerve. The latency of the IPSP was 0.7-0.9 ms longer than that of the EPSP. Because the experiments were conducted at room temperature, 0.7-0.9 ms may account for an extra synaptic delay. The IPSP is most likely mediated by a disynaptic circuit, namely a feed-forward inhibitory circuit.
Synaptic responses of interbarrelette cells
Interbarrelette cells (n = 12) responded to TrV stimulation with a long-lasting EPSP. A small IPSP was superimposed on the peak of the EPSP (top trace of Fig. 6A). The IPSP was reversed at hyperpolarized potential (bottom trace of Fig. 6A) and blocked by bicuculline (10 µM, Fig. 6B, n = 6). Additional application of D-APV (50 µM) blocked the late components of the EPSPs, which were induced by either weak (top traces) or strong stimulation (bottom traces in Fig. 6C, n = 3). A progressive increase of stimulus intensity caused a stepwise increase in EPSP amplitude. This could be observed in the presence (Fig. 6D) or absence (Fig. 7) of bicuculline. Stimulation of TrV at different sites or lower intensity induced an IPSP alone in interbarrelette cells (Fig. 7A, n = 3). Along with a progressive increase in stimulus intensity, the IPSP gave way to a long-lasting EPSP (Fig. 7, B-F). As shown in Fig. 7G, an increase in stimulus intensity produced a stepwise increase in EPSP amplitude and eventually masked the IPSP. From the number of steps, we can estimate that a single interbarrelette cell receives excitatory inputs from at least five to seven trigeminal nerve fibers (n = 6). In response to different stimulus intensities, the latency of the EPSP remained constant (Figs. 6D and 7G). This suggests that interbarrelette cells receive monosynaptic excitation from whisker afferents. The latency of the IPSP was about one synaptic delay (0.7-0.9 ms) longer than the EPSP. Thus the IPSP is mediated by a disynaptic circuit. In three interbarrelette cells, an increase in stimulus intensity evoked another polysynaptic EPSP (Fig. 7, C and H). When strong stimulus intensities were used, the late polysynaptic EPSP merged with the monosynaptic EPSP (Fig. 7G).
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DISCUSSION |
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Previous studies documented morphological features of projection
cells and interneurons of the adult rat brain stem trigeminal complex
and correlated them with peripherally driven response characteristics
(Arends and Jacquin 1993; Jacquin et al.
1986
, 1988
, 1989a
,b
;
Jacquin and Renehan 1995
). However, little is known about the nature of synaptic transmission during a time when brain stem
trigeminal neurons are consolidating whisker-specific patterns. In
recent years, neural activity, via NMDA receptors have been noted as a
major player in whisker-specific pattern formation in the trigeminal
brain stem. Gene deletion or transgenic alterations of receptor subunit
function studies have underscored the role of NMDA receptors in this
process (Iwasato et al. 1997
; Kutsuwada et al.
1996
; Li et al. 1994
). NMDA receptors are
abundant in the trigeminal brain stem complex during pattern formation
(Iwasato et al. 1997
; Rema and Ebner
1996
). The present study shows that both barrelette and
interbarrelette cells receive trigeminal input mediated by NMDA and
non-NMDA receptors. In addition, for barrelette cells, the NMDA
receptor-mediated EPSP is masked by a GABAA
receptor-mediated IPSP. Inhibitory synaptic responses and intrinsic
membrane properties of neurons are thought to play a crucial role in
pattern formation (Erzurumlu and Guido 1996
). These
properties reflect the manner in which pattern forming cells respond to
their synaptic inputs and how they relay pattern-related information to
neurons leading to the somatosensory cortex.
Electrophysiological recordings in adult rat brain stem trigeminal
nuclei show that barrelette cells are unique in their function, such
that they have only single whisker receptive fields. Other relay
neurons and interbarrelette cells respond to stimulation of multiple
whiskers (Jacquin et al. 1986, 1988
,
1989a
,b
; Shipley 1974
). Studies combining
retrograde labeling techniques and immunohistochemistry document that
different classes of BSTC neurons can be distinguished from one another
by their expression of parvalbumin, calbindin, and GABA
(Bennett-Clarke et al. 1992
; Haring et al.
1990
). On the other hand, hardly anything is known about these
cells during the sensitive period for pattern formation. In this study
we found that in the PrV of 4- to 12-day-old rat pups three different
cell types can be readily identified: barrelette, interbarrelette, and
GABAergic cells (see also Arends and Jacquin
1993
; Ginestal and Matute 1993
;
Ma 1991
, 1993
). We further show that
morphologically identified barrelette cells have a prominent
IA and interbarrelette cells have a
prominent IT. Thus we can identify cell
types in the PrV by their active conductances during the first
postnatal week. Ongoing studies are aimed at defining
electrophysiological properties of PrV neurons during earlier
developmental periods.
Based on our current observations, we propose the following neuronal
circuitry for the early postnatal PrV (Fig.
8). Barrelette cells exhibit a
monosynaptic EPSP followed by a disynaptic IPSP after stimulation of
the TrV. Because the stimulation threshold of the EPSP and that of the
IPSP are about the same, they may originate from the same trigeminal
input. The disynaptic IPSP must be mediated by a feed-forward
inhibitory circuit as shown in Fig. 8 (whisker 1 pathway). GABAergic
cells in the PrV most likely serve as inhibitory interneurons in this
feed-forward circuit. An IPSP without the preceding EPSP can be evoked
in barrelette cells by stimulating different sites in the TrV,
suggesting that there is a separate inhibitory circuit other than the
feed-forward inhibitory circuit. In addition, barrelette cells seem to
receive more inhibitory inputs than excitatory ones. Collectively,
these results suggest that some trigeminal fibers only activate the inhibitory circuits that feed to a given barrelette cell. Because the
IPSP has a disynaptic latency, it is most likely mediated via lateral
inhibitory circuitry. Namely, activation of one pathway produces
inhibition in other parallel pathways. The organization of dendritic
trees, and receptive fields of the barrelette cells indicate that each
cell receives excitatory afferent inputs mainly from a single whisker.
Because the TrV fibers are topographically organized (Bates and
Killackey 1985; Erzurumlu and Jhaveri 1992
; Erzurumlu and Killackey 1983
), we can deduce that
barrelette cells receive disynaptic lateral inhibition from neighboring
whiskers (Fig. 8, whisker 2 pathway). Both feed-forward and lateral
inhibition must play a crucial role in determining spatial and temporal
response properties of barrelette cells. The lateral inhibition from
neighboring whiskers sharpens the receptive field of a given barrelette
cell. Most likely, this provides barrelette cells with a
center-surround receptive field organization, similar to that seen in
the retina (Cook and McReynolds 1998
). The IPSP with a
delay of 0.7-0.9 ms curtails the preceding EPSP, so that the
excitatory response in barrelette cells becomes phasic (short-lasting).
The duration of the IPSP may be modulated by both
IA (to prolong) and
IH (to shorten), so that the IPSP is kept at
a constant duration (~150 ms in our case). Such control may be
critical for the temporal resolution of responses along the
trigeminothalamic pathway (Hartings and Simons 1998
).
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A stepwise increase in EPSP (or IPSP) amplitude evoked by increased
stimulus intensity has been observed in other brain structures. It most
probably indicates successive recruitment of additional fibers and
therefore an increase in the number of active synapses (Allen et
al. 1977; Bartlett and Smith 1999
; Mock
et al. 1997
). Although this approach cannot provide the exact
number of afferent fibers, it serves as a useful means to estimate
afferent convergence. For barrelette cells, there appears to be a
greater convergence in the inhibitory circuit than in the excitatory
circuit. In contrast, for interbarrelette cells there seems to be
greater convergence in the excitatory circuit than in the inhibitory
circuit. Most probably, interbarrelette cells receive monosynaptic
excitation from multiple whiskers (Fig. 8, whisker 1 and 3 pathways).
This is in accord with other studies showing that the receptive field of interbarrelette cells are much larger than those of barrelette cells
(Jacquin et al. 1986
, 1988
,
1989a
,b
; Shipley 1974
). These cells may
also receive inputs from other barrelette and/or interbarrelette cells.
Although interbarrelette cells receive a weak lateral inhibition, the
convergence of excitatory inputs reflects the level of activation of
the PrV. Because interbarrelette cells project to other subnuclei within the BSTC (Jacquin et al. 1990
; Nasution
and Shigenaga 1987
), their main function may be to coordinate
the activity of different brain stem trigeminal subnuclei, and to
modulate transmission of afferent inputs to a variety of projection
sites. In a recent study Sandler et al. (1998)
reported
bursting cells with an elongated dendritic field and
Ni2+-blocked (most likely T-type) Ca2+
conductance in the PrV of the gerbil. Although this study did not note
the location of the cells recorded from in the PrV, these cells appear
similar to morphologically identified interbarrelette cells described
in the present study.
Within the rodent BSTC, PrV barrelette cells are the key players in
conveying whisker-related patterns to the dorsal thalamus (Killackey and Fleming 1985). However, in two subnuclei
of the spinal trigeminal nucleus (subnuclei interpolaris and caudalis), whisker-related patterns, i.e., barrelettes, are also present. In fact,
the most conspicuous barrelettes of the BSTC are found in the
subnucleus interpolaris (SPI). Recent work from our laboratory showed
that IH, IT, and
IA are present in SPI neurons as well (Guido et al. 1998
). However, we have not yet correlated
these conductances with morphologically identified specific cell types. Therefore we do not know whether barrelette and interbarrelette cells
of the SPI also exhibit class-specific conductances like the cells of
the PrV.
Collectively, our results from studies on the PrV and SPI suggest that unique combinations of electrophysiological properties of the developing BSTC neurons could enhance activity-dependent modeling of neural connections during pattern formation. Electrophysiological properties of barrelette cells are especially suited for detection of patterned trigeminal inputs from the whiskers and the transmittal of pattern-related information to the barreloids of the dorsal thalamus with sharpened receptive field and phasic temporal properties. Interbarrelette cells with "diffuse"projections to other upstream trigeminal-recipient brain regions sample excitatory inputs from multiple whiskers and barrelette cells.
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ACKNOWLEDGMENTS |
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We thank T. Ulupinar for help with histology and manuscript preparation and Dr. E. Ulupinar for help with immunohistochemistry.
This project was supported by the Whitehall Foundation and by National Institute of Neurological Disorders and Stroke Grant NS-37070 to R. S. Erzurumlu.
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FOOTNOTES |
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Address for reprint requests: F.-S. Lo, Dept. of Cell Biology and Anatomy, Louisiana State University Medical Center, 1901 Perdido St., New Orleans, LA 70112.
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 2 February 1999; accepted in final form 23 June 1999.
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REFERENCES |
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