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1 Department of Molecular and Integrative Physiology, University of Illinois, Urbana, Illinois 61801; 2 Department of Pharmacology, University of Illinois, Urbana, Illinois 61801
Submitted 24 March 2003; accepted in final form 11 April 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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;
Huguenard and Prince 1994;
Steriade et al. 1993;
von Krosigk et al. 1993;
Warren et al. 1994). These
rhythms are correlated not only with the level of arousal but are also
observed in certain pathophysiological states such as generalized absence
epilepsy (Domich et al. 1986;
Steriade and Llinás
1988; Steriade et al.
1993; Williams
1953). A variety of neuromodulators, such as acetylcholine (ACh),
norepinephrine (NE), or serotonin (5HT), which arise from brain stem nuclei,
can attenuate these intrathalamic rhythms (Lee and McCormick
1996,
1997;
McCormick 1992). In addition
to these brain stem-derived compounds, another class of modulators, namely the
neuropeptides, have been localized within the thalamocortical circuit and may
also play an important role in the regulation of intrathalamic rhythmic
activities (Cox et al. 1997;
Lee and McCormick 1997;
Sun et al. 2002). The
neuropeptides are of interest because these compounds have been found to be
co-localized with classical neurotransmitters [e.g., 
-aminobutyric acid
(GABA) and glutamate], released in an activity-dependent manner, and produce
long-lasting changes in neuronal excitability in other brain regions
(Bartfai et al. 1988;
Buijs et al. 1995;
Christenson et al. 1991;
Hendry et al. 1984;
Lundberg and Hökfelt
1983; Stamp and Semba
1995).
A variety of neuropeptides and their receptors are localized within the
thalamus (Baldino et al. 1989;
Burgunder and Young 1988;
Doetsch et al. 1993;
Graber and Burgunder 1996;
Kaneko and Mizuno 1988;
Lanaud et al. 1989;
Molinari et al. 1987;
Staun-Olsen et al. 1985). The
functional significance of these modulators within thalamic nuclei has
remained somewhat elusive. However, recent studies have scratched the surface
regarding peptide actions in the thalamus, demonstrating that these compounds
may alter passive membrane characteristics of thalamic neurons such as resting
membrane potential, input resistance, and membrane conductance, and in some
cases, such actions can modulate intrathalamic rhythmic activities (Cox et al.
1995,
1997;
Leresche et al. 2000;
Sun et al. 2001).
Vasoactive intestinal peptide (VIP) is a 28-amino-acid peptide that was
first isolated from porcine intestine and is involved with many regulatory
functions, including vasodilation, gastrointestinal secretion and motility,
and glycogenolysis (Gozes and Brenneman
1989). VIP is also widely distributed throughout the central and
peripheral nervous system where it may serve as a putative neuromodulator
(Gozes and Brenneman 1989).
VIP has been found to produce a variety of actions including alterations in
intrinsic properties of neurons or synaptic transmission in the CNS
(Gozes and Brenneman 1989;
Kohlmeier and Reiner 1999;
Murphy et al. 1993;
Wang et al. 1997). This
peptide has been localized within TRN neurons
(Burgunder et al. 1999b), and
VIP receptors are distributed within primary relay thalamic nuclei such as VB
and the dorsal lateral geniculate nucleus
(Sheward et al. 1995;
Usdin et al. 1994;
Vertongen et al. 1997). The
actions of VIP within this brain region remain unknown and are the focus of
this study. Considering that intrathalamic rhythmic activity requires short,
high-frequency discharges of thalamic neurons and this condition may be
optimal for peptide release (Bartfai et al.
1988), we speculate that VIP may serve as an endogenous regulator
of intrathalamic rhythms. In this study, we investigated the actions of VIP on
intrathalamic rhythmic activities as well as the actions of this peptide on
the excitability of neurons at the single cell level. Our results indicate
that VIP strongly attenuates slow (2–4 Hz) intrathalamic rhythmic
activity. In addition, VIP selectively enhances a hyperpolarization-activated
cation current, Ih, in relay neurons, but produces
negligible effects in TRN neurons. Some of these findings have been presented
in abstract form (Lee and Cox
2002).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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). Young Sprague-Dawly rats (postnatal age 9–18 days)
were deeply anesthetized with pentobarbital sodium (50 mg/kg) and decapitated.
The brain was quickly removed and placed into cold, oxygenated slicing medium
containing (in mM) 2.5 KCl, 10.0 MgCl2, 0.5 CaCl2, 1.25
NaH2PO4, 26.0 NaHCO3, 11.0 glucose, and 234.0
sucrose. Tissue slices (300–500 µm) were cut in the horizontal plane
using a vibrating tissue slicer, transferred to a holding chamber, and
incubated 
1 h before recording. Individual slices were then transferred to
a recording chamber and continuously superfused with oxygenated physiological
saline at 30°C. The physiological solution used in the experiments
contained (in mM) 126.0 NaCl, 2.5 KCl, 1.25 MgCl2, 2.0
CaCl2, 1.25 NaH2PO4, 26.0 NaHCO3,
and 10.0 glucose. This solution was gassed with 95% O2-5%
CO2 to a final pH of 7.4.
Intracellular recordings, using the whole cell configuration were obtained
with the visual aid of a modified Axioskop 2FS equipped with infrared
differential interference contrast optics (Zeiss Instruments, Thornwood NY).
Recording pipettes were pulled from 1.5 mm OD capillary tubing using a
two-stage pipette puller and had tip resistances of 3–6 M
when
filled with the following intracellular solution (in mM) 117 K-gluconate, 13
KCl, 1.0 MgCl2, 0.07 CaCl2, 0.1 EGTA, 10.0 HEPES, 2.0
Na2-ATP, and 0.4 Na-GTP. The pH was adjusted to 7.3 using KOH and
osmolarity was adjusted to 290–300 mosM with distilled H2O.
The initial access resistance typically ranged from 10 to 25 M and
remained stable during most recordings included for analyses in this
study.
For intracellular recordings, individual slices were transferred to a submersion-type recording chamber on a modified microscope stage. A low power objective (x5) was used to identify various thalamic nuclei and a high-power water immersion objective (x63) was used to visualize individual neurons. An Axoclamp2B amplifier (Axon Instrument, Foster City, CA) was used in bridge mode for voltage recordings or switching single-electrode voltage-clamp mode for current recordings. Voltage and current protocols were generated using pClamp software (Axon Instruments), and data were stored on an IBM PC-compatible computer. For current-clamp recordings, an active bridge circuit was continuously adjusted to balance the drop in potential produced by passing current through the recording electrode. The apparent input resistance was calculated from the linear slope of the voltage-current relationship obtained by applying constant current pulses ranging from –100 to +40 pA (800 ms duration). During VIP application, change in input resistance was determined by membrane response to single-intensity constant current hyperpolarizing pulses (5–40 pA, 500 ms, 0.2Hz).
For voltage-clamp recordings, an Axoclamp2B amplifier (Axon Instruments)
was used in discontinuous mode. In these recordings, the switching frequency
ranged from 2.5 to 3.5 kHz with a gain of 150–800 pA/mV, and the
headstage was continually monitored to ensure that the current transients have
completely decayed before voltage measurements. Voltage-clamp recordings were
limited to neurons that had a stable access resistance <30 M. To
quantify VIP-mediated changes in membrane conductance, slow ramped voltage
commands (-60 to –110 mV, 4 s duration, 0.1 Hz) were applied to the
neuron. For analyses, three subsequent current traces were averaged prior to
VIP application and at peak changes produced by VIP.
Extracellular multiple-unit recordings were obtained using sharpened
tungsten microelectrodes (1–4 M; Frederick Haer, Bowdoinham, ME).
All data were digitized (1–2 kHz) and stored using Axotape software
(Axon Instruments). Monopolar electrical stimulation was applied to either TRN
or internal capsule using sharpened tungsten electrodes (200–600
k, Frederick Haer).
Concentrated stock solutions of VIP (0.3 mM) were prepared in distilled
water and diluted in physiological saline to a final concentration of
0.05–3.0 µM. VIP was applied by injecting a bolus into the input line
of the chamber over 60 s using a motorized syringe pump. Based on the rate of
VIP injection and the rate of chamber perfusion, the final bath concentration
of VIP was estimated to one-eighth of the concentration introduced in the flow
line (Cox et al. 1995).
Control injections of physiological saline produced neither changes in
intrathalamic activity during extracellular recording or changes in membrane
potential or input resistance during current-clamp recordings, suggesting that
the temporary increase in flow rate during the bolus injections had no effect
on the recordings. All antagonists were bath applied in final concentration.
VIP was purchased from Calbiochem (San Diego, CA) and
N-ethyl-1,6-dihydro-1,2-dimethyl-6-(methylimino)-N-phenyl-4-pyrimidin
amine (ZD7288) from Tocris (Ellisville, MO). All remaining compounds were
purchased from Sigma (St. Louis, MO).
Analyses of intrathalamic rhythmic activities were similar to those
described previously (Cox et al.
1997). Briefly, autocorrelograms were constructed from the
extracellular multiple-unit data over a period of 3–9 s with a bin size
of 30 ms to quantify degree of synchrony and duration of intrathalamic
oscillations (Minianalysis, Synaptosoft, Leonia, NJ). Three measures were used
to quantify oscillatory activity in autocorrelograms: number of peaks,
oscillation amplitude (Amposc), and frequency of oscillation.
Alterations in Amposc suggest a change in the number of units
participating in the rhythmic activity. The number of peaks indicates the
number of cycles in the rhythm, and the oscillation frequency reflects the
principle frequency of the rhythmic activity. All data are presented as mean
± SD, except where noted. Most statistical analyses consist of
Mann-Whitney U test and, when appropriate, the Wilcoxon test for
paired samples. In some noted instances, a paired Student's t-test
was used for testing statistical significance. The difference between the
means was considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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The reciprocal synaptic connectivity between thalamic reticular nucleus
(TRN) and adjacent ventrobasal nucleus (VB), which is preserved in the in
vitro slice preparation, supports intrathalamic rhythmic activities
(Fig. 1A)
(Cox et al. 1997;
Huguenard and Prince 1994;
von Krosigk et al. 1993;
Warren et al. 1994).
Electrical stimulation in TRN or adjacent internal capsule (IC) typically
evoked an arrhythmic discharge that could last 
2 s
(Fig. 1Bi). In a few
remote cases, rhythmic discharge ranging from 3.2 to 6.0 Hz was observed in
control artificial cerebrospinal fluid (ACSF; n = 3; data not shown).
However, after addition of the GABAA receptor antagonist, BMI (10
µM), electrical stimulation produced a stable rhythmic activity ranging
from 2.2 to 3.3 Hz that could last many seconds
(Fig. 1Bii)
(Bal et al. 1995b;
Cox et al. 1997). Under these
conditions, the frequency and duration of the rhythmic activity was very
stable from trial to trial.
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We next tested the effects of VIP on this slow, intrathalamic rhythmic
activity in 27 slices. Bath application of VIP (0.1–1.0 µM, 60-s
duration) dramatically suppressed the rhythmic oscillations in 18 of 27 slices
tested. As illustrated in Fig.
2Ai, VIP (0.75 µM) strongly attenuated the slow
rhythmic activity. The contour plot (Fig.
2Bi) illustrates that the maximum effect occurred 120 s
after VIP treatment, and recovered near predrug levels 
5 min after VIP
application. In addition to the suppression of the rhythmic activity, the
intraburst frequency was increased from 2.4 to 2.8 Hz after VIP application,
and a similar shift in frequency (>10%) was observed in 4 of 18 slices
mentioned in the preceding text. The autocorrelogram clearly indicates a
highly synchronized response that lasts eight cycles (3 s) in control
condition (Fig. 2Ci,
black line). In VIP the number of cycles, as well as the total number of
spikes per episode was reduced (Fig.
2Ci, orange line).
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The effects of VIP on the rhythmic activity appeared concentration dependent despite variability of the response from slice to slice. As illustrated in Fig. 2, Aii–Cii, a lower VIP concentration (0.2 µM) did not alter the intrathalamic oscillation. Similarly, the lower VIP concentrations (0.1–0.2 µM) attenuated the rhythmic activity in only five of nine slices. At relatively higher VIP concentrations (0.3–1.0 µM), the intrathalamic rhythmic activity was attenuated in 14 of 18 slices. The rhythmic activity typically returned to control conditions within 5 min after VIP application. The effects of varying VIP concentration on the rhythmic activity are illustrated in Fig. 2D. From the autocorrelograms we quantified the number of peaks, Amposc, and intraburst frequency. These different measures were generally consistent with the overall VIP effect, and reached statistical significance in many instances (P < 0.05; Mann-Whitney U test).
VIP depolarizes thalamic relay neurons
The persistence of the rhythmic activity is dependent on the firing mode of
TRN and relay thalamic neurons, which is closely related to the membrane
potential (Jahnsen and Llinás
1984a; Steriade and
Deschênes 1984; Steriade
and Llinás 1988; von
Krosigk et al. 1993). Because the membrane potential largely
determines the firing mode (i.e., burst- or single-spike discharge), we next
examined the action of VIP on the membrane potential of thalamic neurons.
Intracellular recordings using whole cell configuration were obtained from 46
relay and 8 TRN neurons. The average resting membrane potential of relay
neurons (–64.4 ± 4.2 mV; n = 46) was significantly
different from that of the TRN neurons (–71.4 ± 3.2 mV;
n = 8; P < 0.001). Despite the difference in resting
membrane potentials, the apparent input resistance did not significantly
differ between the relay neurons (173.4 ± 75.8 M; n =
46) and the TRN neurons (198.1 ± 64.2 M; n = 8).
Bath application of VIP (0.5 µM) produced a relatively slow-onset, long-duration depolarization in VB relay neurons (Fig. 3A, top). This VIP concentration (0.5 µM) produced a depolarization in all 18 relay neurons tested, and the amplitude of the response averaged 2.7 ± 0.8 mV (n = 18). The latency to peak of the VIP-mediated depolarization occurred 136 ± 32 s after VIP application. The average duration of the depolarization was 355 ± 88 s for the 12 of 18 neurons that completely recovered to the preVIP membrane potential. In the remaining six neurons, the membrane potential partially recovered to predrug levels. Associated with the VIP-mediated membrane depolarization, there appeared to be a decrease in apparent input resistance (Fig. 3, B and C). Small hyperpolarizing current steps (–5 to –40 pA; 500-ms duration; 0.2 Hz) were used to access changes in input resistance. During the peak of the VIP-mediated depolarization, the membrane potential was manually repolarize to predrug levels to test for voltage-independent changes in apparent input resistance. In 10 relay neurons, VIP was found to produce a small decrease in the input resistance by 12.5 ± 11.2% (n = 10). VIP decreased input resistance by 10.4 ± 13.5% in normal ACSF (n = 6) and by 15.8 ± 7.2% in presence of 1 µM TTX (n = 4). In contrast to VIP-mediated effects on relay neurons, VIP (0.5 µM) produced no detectable change in membrane potential or apparent input resistance in seven of eight TRN neurons (Fig. 3A, bottom). A short-duration depolarization (185 s) was evoked in a single TRN neuron.
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To determine whether the VIP-mediated depolarization was due to a postsynaptic action, we applied VIP in the presence of tetrodotoxin (TTX, 1 µM). VIP was applied in the presence of TTX in 6 of the 18 cells mentioned in the preceding text, and the remaining 12 cells were tested in the presence of control ACSF. We analyzed the initial application of VIP because repeated applications resulted in a decreased response (see following text). Based on the first application the VIP-mediated response in control ACSF averaged 2.7 ± 0.9 mV (n = 12) and 362 ± 110 s duration (n = 8) and did not differ significantly from the VIP-mediated response in the presence of 1 µM TTX [2.7 ± 0.7 mV (n = 6); 343 ± 16 s (n = 4); Fig. 3, B and C]. With repeated VIP applications at 10-min intervals in control conditions, we found that the second response to VIP was attenuated by 26 ± 37% (n = 6) relative to the first VIP-mediated depolarization. A similar decrease in the membrane depolarization (32 ± 24%) was observed in four neurons in which VIP was initially applied in control conditions, and the second VIP application was given in the presence of TTX (1 µM). These data suggest that the VIP-mediated depolarization is likely due to a postsynaptic site of action and, second, that there may be some desensitization of the response to subsequent applications of VIP.
We next determined whether the VIP-mediated depolarization was
concentration dependent. For this experiment, we only included the response to
the initial VIP application because of the desensitization to repeated VIP
applications mentioned in the preceding text. The VIP-mediated depolarization
appears dose dependent in the range of 0.05–3.0 µM
(Fig. 4C). Low VIP
concentration (0.05 µM) produced no detectable change in five of seven
cells but produced small depolarization in the two remaining neurons. An
intermediate VIP concentration (0.5 µM) produced relatively repeatable and
reversible depolarization when applied at 10-min intervals in all relay
neurons tested (2.7 ± 0.8 mV; n = 18). Higher VIP
concentration (1.5 and 3.0 µM) produced larger depolarization (3.8 ±
1.5 mV; n = 12; Fig.
4B, bottom) that was partially reversible in 7
of 12 neurons. As expected, the time to peak of depolarization (109 ±
26 s; n = 9) evoked by high VIP concentration (>1.5 µM) is
significantly shorter than that of depolarization (134 ± 30 s;
n = 29; P < 0.05) evoked by low VIP concentration
(0.5 µM; Fig.
4B, top). Summary of VIP effects on resting
membrane potential of VB neurons is shown in
Fig. 4C. In six
neurons, low-concentration VIP application (0.05 or 0.1 µM) was followed 10
min later by a higher VIP concentration (0.5 µM;
Fig. 4A). Although the
amplitude of the VIP depolarization across cells varied greatly, in five of
six neurons, the greater VIP concentration (2nd application) produced a larger
depolarization (Fig.
4D). This increasing amplitude response to the higher
concentration persisted despite our earlier evidence that some desensitization
may occur with subsequent applications. We speculate that the
concentration-dependence for the response is underestimated in this
experiment. Nonetheless, the response to VIP appears to be concentration
dependent.
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VIP enhances Ih in relay neurons
Voltage-clamp recordings were used to characterize the conductance changes produced by VIP in relay neurons. Similar to our current-clamp recordings in the preceding text, VIP (0.5 µM) produced a reversible inward current that lasted many minutes (Fig. 5A). In control ACSF or TTX (1.0 µM), VIP produced an inward current in all 16 cells tested that averaged 20 ± 8 pA (n = 16). The duration of VIP-mediated inward current averaged 342 ± 62 s, and the time to peak of the inward current was 132 ± 21 s, similar to our current-clamp recordings. We used slow voltage command ramps (–60 to –120 mV, 2-s duration, 0.1 Hz) to determine the voltage characteristics of the conductance change by VIP (Fig. 5B). In the predrug condition, the current response to the ramped voltage command is nonlinear (Fig. 5B, predrug). During the ramped voltage command, there is an increase in conductance at more hyperpolarized potentials, which is likely due in part to activation of the hyperpolarization-activated mixed cation conductance, Ih. To quantify changes in the "resting" conductance of the neuron, we analyzed the initial portion of the current response near resting membrane potential (–60 to –80 mV). The resting conductance of the neurons prior to VIP treatment averaged 4.6 ± 2.2 nS. After application of 0.5 µM VIP, the conductance was significantly increased to 5.7 ± 2.6 nS (n = 14; P < 0.02, Wilcoxon test). Of the 14 cells tested, 5 of them were in control ACSF, and the remaining 9 in 1.0 µM TTX. The increased conductance by VIP did not differ between these two conditions (P > 0.9), and thus these data have been combined. VIP increased membrane conductance in 11 of 14 relay neurons but had no effects on membrane conductance in the remaining 3 neurons. The increased conductance by VIP returned to baseline levels in all 11 neurons. To access the voltage dependence of the conductance altered by VIP, we next subtracted the difference of the current responses before and after VIP application (Fig. 5C). This "VIP-mediated conductance" (Idiff), was usually linear over the voltage range of –60 mV to –90/–100 mV. Extrapolating the linear fit of Idiff indicated that the conductance sensitive to VIP had a reversal potential of –45 ± 6 mV (n = 10, range –57 to –35 mV; Fig. 5C).
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Considering the reversal potential of VIP-mediated current in relay neurons
(–45 ± 6 mV) is similar to the reported reversal potential of
Ih in relay neurons
(McCormick and Pape 1990b;
Zhu et al. 1999) and the
steepest slope of membrane conductance altered by VIP appears to be between
–80 and –100 mV, a voltage range at which Ih
is strongly activated, we speculated that VIP may alter Ih
in the relay neurons. We tested the possible action of VIP receptor activation
on Ih by using the Ih antagonist, ZD7288 in
five relay neurons. As illustrated in Fig.
6Ai, VIP produced an inward current in the presence of
TTX. After bath application of ZD7288 (100 µM), the subsequent application
of VIP produced no apparent change in the holding current. A similar
observation was made for all five neurons tested. That is, VIP produced an
inward current that averaged 22 ± 6 pA in TTX and after application of
ZD7288, VIP application produced a significantly smaller inward current that
averaged 5 ± 7 pA (P < 0.05 paired t-test).
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In three of the five neurons, VIP produced an increase in membrane conductance (35 ± 18%; e.g., Fig. 6A). The VIP-mediated current had an extrapolated reversal potential of –45 mV (Fig. 6Aiii, left). In the presence of ZD7288 and TTX, VIP produced a smaller increase in membrane conductance (10 ± 8%, n = 3, Fig. 6A, ii and iii, right). As illustrated in Fig. 6Aiii, the VIP-sensitive current was completely attenuated in the presence of ZD7288. In the remaining two neurons VIP produced an inward current but did not produce detectable changes in membrane conductance in the range of –60 to –80 mV, the range that we had used to quantify conductance changes (Fig. 6B, i and ii, left). A steep-sloped VIP-sensitive current was present at more hyperpolarized potentials, and in the presence of ZD7288, this current was completely attenuated (Fig. 6B, i and ii, right). The lack of response in the –60- to –80-mV range was likely due to space-clamp error arising from recording of intact neurons with extensive intact dendritic processes. Nonetheless, the pharmacological manipulation using ZD7288 is consistent with an effect on Ih by VIP.
The sensitivity of the VIP-mediated responses to the antagonist ZD7288 suggests that VIP may modulate/activate Ih. To investigate the possible action of VIP on Ih, we used long-duration (2.4 s) voltage step commands (–50 to –120 mV; 10-mV increment; 0.125 Hz) to evoke Ih (Fig. 7A, V). The current responses to the step commands consisted of an initial instantaneous response followed by a slow inward current that typically reached a steady state after a couple of seconds (Fig. 7A, I). We calculated the difference between the initial instantaneous response (Iins) and the steady-state level (Iss) as a measure of Ih. In control conditions, there is little Ih in response to the initial small voltage commands, but with more hyperpolarizing voltage commands, there is an increase in the slow inward current (Fig. 7A, black line). After VIP application (0.5 µM; Fig. 7A, gray line), there appears to be little change in the smaller voltage commands (–60 to –70 mV); however, there is an obvious increase in the mid-ranged voltage commands (–80 to –110 mV). This increase in apparent Ih is quite obvious in our population data based on four neurons (Fig. 7B). To quantify this increase in Ih activation by VIP, we fit each activation curve (pre-VIP and VIP) using a sigmoid function. VIP produced an average 5.2 ± 0.4 mV shift in the depolarizing direction, and this alteration was statistically significant (n = 4; P < 0.05). These data suggest that at more depolarized voltage commands at which Ih is inactivated, VIP should produce less inward current. In four neurons, we initially applied VIP to cells with a Vhold = –50 mV, and the resulting inward current averaged 5.3 ± 1.0 pA, which was within our noise level. If the Vhold was then changed to either –70 or –80 mV, and VIP was applied again, the resulting VIP-mediated inward current averaged 18.3 ± 5.4 pA, significantly larger than that with the depolarized Vhold (P < 0.02, paired t-test). These data indicate a voltage dependence of the VIP effect and that the VIP enhances a basal Ih, but probably does not activate Ih directly.
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To investigate the time course of this alteration in Ih, we also employed single voltage step command (–50 to –80 mV; 2-s duration; 0.1 Hz; n = 4) to evoke Ih (Fig. 7C, V). The amplitude of Ih prior to VIP application averaged 14.2 ± 7.6 pA. After VIP (0.5 µM) application, the amplitude of Ih average 32.8 ± 14.3 pA, significantly greater than that of preVIP levels (Fig. 7C, I; P < 0.02, paired t-test). The time course of the VIP effect on Ih closely approximated the duration of the VIP-mediated depolarization (Fig. 7D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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; Kupfermann
1991), our knowledge regarding their functional role in the
mammalian CNS is limited. Considering these intrathalamic rhythmic activities
are associated with behavioral states as well as some pathophysiological
conditions such as generalized absence epilepsy
(Huguenard and Prince 1994;
Steriade and Llinás
1988; Steriade et al.
1993; von Krosigk et al.
1993), we speculate that VIP may play a potentially important role
in the regulation of such activities. Our results indicate that VIP
selectively depolarized thalamic relay neurons but had negligible actions on
TRN neurons. This depolarization results from an enhancement of the
hyperpolarization activated cation conductance, Ih. The
resulting depolarization may in turn decrease the potential burst output of
thalamic relay neurons and as a result attenuate the intrathalamic rhythmic
activity. Our working hypothesis is that small amounts of VIP are released
during each cycle of the rhythmic activity and thus its concentration
accumulates with each cycle. As VIP eventually reaches threshold of its action
on relay neuron, it may then in turn decrease the burst firing discharge of
these cells and thereby attenuate the rhythmic activity. VIP depolarizes relay neurons in VB by enhancing Ih
Our results demonstrate that VIP selectively depolarized all relay neurons
tested with no apparent effect on TRN neurons. Although neurophysiological
studies regarding the actions of VIP are few, this peptide has been found to
produce a number of different actions within the CNS. VIP has been found to
depolarize brain stem neurons and increase the excitability of hippocampal and
neocortical neurons (Haas and Gahwiler
1992; Kohlmeier and Reiner
1999; Murphy et al.
1993; Pawelzik et al.
1992). This peptide has also been shown to enhance GABAergic
synaptic transmission in cultured hippocampal neurons presumably through a
presynaptic site of action (Wang et al.
1997). As with most other transmitter systems, multiple subtypes
of VIP receptors have been identified, namely VPAC1 and
VPAC2, (Gozes and Brenneman
1989; Harmar et al.
1998). The messenger RNAs (mRNAs) for these two receptor subtypes
are differentially distributed in the CNS. The mRNA encoding the
VPAC1 receptor is most abundant in the cerebral cortex and
hippocampus (Ishihara et al.
1992). In contrast, the highest concentration of mRNA encoding the
VPAC2 receptor is found in the thalamus and suprachiasmatic nucleus
(Sheward et al. 1995;
Usdin et al. 1994). Thus we
predict that the VIP-mediated actions we have observed in VB neurons is likely
mediated via a VPAC2 receptor, but the lack of potent, selective
antagonists have prevented a direct testing of this hypothesis.
Based on our physiological and pharmacological data, the depolarization of
VB neurons produced by VIP results from an enhancement of
Ih in the relay neurons. Our study indicates that the
reversal potential of the current evoked by VIP is consistent with the
reversal potential of Ih
(McCormick and Pape 1990b;
Zhu et al. 1999). Second, the
Ih blocker ZD7288 completely attenuated the VIP
receptor-mediated inward current and accompanying alterations in membrane
conductance. Finally, the VIP-mediated response appears voltage dependent.
That is, at more depolarized holding potentials in which most
Ih channels are inactivated
(McCormick and Pape 1990b;
Zhu et al. 1999), the
VIP-mediated response is within noise levels. In contrast, the largest effect
of VIP is in the –80- to –100-mV voltage range in which
Ih is strongly activated. A similar enhancement of
Ih by NE and 5HT has also been demonstrated in thalamic
relay neurons (McCormick and Pape
1990a). A common aspect of these modulators is their ability to
engage particular intracellular second-messenger pathways, namely cyclic AMP.
Recent studies have demonstrated that the direct binding of cyclic AMP to the
cytoplasmic site on Ih channels can enhance the rate of
Ih activation (Beaumont
and Zucker 2000; Wainger et
al. 2001). Because VIP receptors are linked to G-proteins that
activate adenylate cyclase (Hashimoto et
al. 1993; Lutz et al.
1993), we predict that cytosolic concentration of cyclic AMP would
increase after activation of VIP receptors. Thus the alteration in cyclic AMP
activity may be the common effector mechanism by which these different
modulators (i.e., VIP, NE, and, 5HT) exert their actions. The functional
significance of these different modulators may be due to the origin of these
compounds. NE and 5HT arise from brain stem sources whose activity is
dependent on the behavioral level of the animal
(Aston-Jones and Bloom 1981;
Foote and Morrison 1987;
Trulson and Jacobs 1979). In
contrast, VIP is localized within TRN neurons
(Burgunder et al. 1999a) and
thus VIP release would be dependent on activity of the thalamocortical
circuit.
Functional significance of VIP effects on intrathalamic oscillation
Intrathalamic rhythmic activities are closely related to animals'
behavioral states, as well as certain pathophysiological conditions such as
generalized absence epilepsy (for review, see
Domich et al. 1986;
Steriade and Llinás
1988; Steriade et al.
1993; Williams
1953). The reciprocal synaptic connection between TRN (or
analogous perigeniculate nucleus in visual system) and relay nuclei provides
the anatomical organization for intrathalamic oscillations. Another key factor
for rhythmic activity involves intrinsic membrane properties of thalamic
neurons that will ultimately determine the firing mode of these cells: burst
or tonic-firing mode. Certain neuromodulators, such as ACh, NE, or 5HT, which
arise from brain stem nuclei, alter the firing modes of thalamic neuron and
thereby attenuate these rhythms (Lee and McCormick
1996,
1997). Other modulators, that
may to be intrinsic to the thalamic circuitry such as cholecystokinin (CCK)
and somatostatin (SS) have also been found to alter the firing mode of
thalamic neurons and attenuate intrathalamic rhythmic activity
(Cox et al. 1997;
Sun et al. 2002). Our data
suggest that a different endogenous modulator, VIP can strongly suppress
intrathalamic oscillation. VIP is contained within TRN neurons whose output is
restricted within the thalamus (Burgunder
et al. 1999a) and thus may serve as an endogenous modulator of
thalamic circuit. Thus understanding the variety of modulators that may
regulate the firing mode of these cells will ultimately provide a better
understanding of the conditions that give rise to the initiation or cessation
of the intrathalamic rhythmic activities.
The fundamental mechanisms required to maintain the intrathalamic rhythmic
activity are well understood (Bal et al.
1995a,b,
2000;
Huguenard and Prince 1994;
Steriade and Deschênes
1984; Steriade and
Llinás 1988; Steriade
et al. 1993; von Krosigk et
al. 1993). While there are a number of key characteristics
required for the rhythmic activity, a critical aspect is the firing mode of
thalamic neurons. Thalamic neurons discharge action potentials in two basic
modes: tonic and burst firing (Jahnsen and Llinás
1984a,b;
Steriade and Deschênes
1984). The occurrence of either of these firing modes is dependent
on voltage-dependent intrinsic properties of these neurons and ultimately
depends on the activation of low-threshold, voltage-dependent
Ca2+ current, IT. At relatively
depolarized membrane potentials, IT is inactivated and the
cell responds in tonic mode in which the output of the cell is linearly
related to the strength of depolarization. In contrast, at relatively
hyperpolarized membrane potentials, channels underlying IT
are deinactivated, and with activation of IT, the neuron
fires a short-duration (100–200 ms), high-frequency (>200 Hz)
discharge of action potentials. To maintain the rhythmic activity, cells in
relay nuclei as well as TRN must discharge in burst mode (Bal et al.
1995a,b;
Steriade and Deschênes
1984). Therefore modulators that alter the firing mode of these
neurons, attenuate the rhythmic activity.
Numerous neuromodulators, including ACh, glutamate (via metabotropic
receptors), NE, 5HT, CCK, and histamine depolarize either relay neurons or
TRN/PGN (perigeniculate nucleus) neurons by decreasing the resting leak
potassium current, shifting the discharge mode of these neurons into a tonic
mode and ultimately terminating the rhythmic activity
(Cox et al. 1997; Lee and
McCormick 1996,
1997;
McCormick and Williamson
1991). In addition, NE, 5HT, and histamine have been found to
enhance Ih leading to membrane depolarization of varying
amplitudes and ultimately terminating the intrathalamic oscillation
(Lee and McCormick 1996;
McCormick and Pape 1990a;
McCormick and Williamson
1991). It has been suggested that the increase in membrane
conductance associated with increased Ih can attenuate the
effectiveness of inhibitory synaptic activity and lead to a decreased
probability of burst discharge from relay neurons
(Bal and McCormick 1996;
McCormick and Pape 1990a). Our
results indicate that VIP enhances Ih and thus may share a
common effector mechanism as described for NE and 5HT. The possible
attenuation of inhibitory activity by VIP may be the dominant action of VIP
considering that the overall membrane depolarization produced by VIP is only a
few millivolts and may not be of the magnitude expected to clearly shift these
relay neurons from burst to tonic firing mode. However, our extracellular data
clearly indicate that VIP produces an extremely strong attenuation of the
rhythmic activity but additional experiments are necessary to investigate the
influence of VIP on inhibitory activity within the thalamus.
Although various neuromodulators may share common effector mechanisms, an
obvious difference among them is their origin. Many of these compounds studied
thus far, ACh, NE, 5HT, and histamine, originate from brain stem regions, and
thus their activity is closely associated with levels of arousal and
sleep-wake states (McCormick and Bal
1997; Steriade et al.
1993). The neuropeptides studied thus far, VIP, CCK, SS, and
neuropeptide Y, are endogenous to the intrathalamic circuit, and therefore the
release of these compounds is closely correlated to intrathalamic circuit
activity. Considering high-frequency spike discharges may be optimal to
release peptide (Bartfai et al.
1988), we speculate that the influence of VIP might be most
prominent during this rhythmic activity or during other conditions in which
TRN neurons discharge at high frequencies. We speculate that burst discharge
of TRN neurons produces the release of VIP, and with each cycle, the actions
of VIP would summate on VB neurons. VIP receptor-mediated depolarization
associated with increase in membrane conductance by enhancing
Ih would bias the firing mode of the thalamic relay
neurons toward a tonic mode and thereby attenuate the intrathalamic rhythmic
activity. VIP-mediated attenuation of intrathalamic oscillation may be
associated with alteration of behavioral state and imbalances in this
peptidergic system may predispose this network to increased rhythmic activity
as observed in certain pathophysiological conditions such as generalized
absence epilepsy.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests: C. L. Cox, Dept. of Molecular and Integrative Physiology, 524 Burrill Hall, 407 S. Goodwin Ave., Urbana, IL 61801 (E-mail: clcox{at}life.uiuc.edu).
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REFERENCES |
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