1Institute for Developmental Neuroscience, Vanderbilt University; 2Institute for Molecular Neuroscience, Vanderbilt University School of Medicine; and 3Department of Psychology, Vanderbilt University, Nashville, Tennessee 37240
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ABSTRACT |
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Sachdev, Robert N. S., Heike Sellien, and Ford F. Ebner. Direct Inhibition Evoked by Whisker Stimulation in Somatic Sensory (SI) Barrel Field Cortex of the Awake Rat. J. Neurophysiol. 84: 1497-1504, 2000. Whisker deflection typically evokes a transient volley of action potentials in rat somatic sensory (SI) barrel cortex. Postexcitatory inhibition is thought to quickly terminate the cortical cell response to whisker deflection. Using dual electrode extracellular recording in awake rats, we describe an infrequent type of cell response in which stimulation of single hairs consistently blocks the ongoing discharge of neurons without prior excitation (I-only inhibition). Reconstruction of the recording sites indicates that I-only inhibition occurs most frequently when the recording site is clearly in the septum or at the barrel-septum junction. The same cells that respond with I-only inhibition to one whisker can show an excitatory discharge to other whiskers, usually followed by inhibition. Stimulation of either nose hairs or the large mystacial vibrissa can evoke I-only inhibition in SI cortex. I-only inhibition is most commonly observed at low stimulus frequencies (~1 Hz). At stimulus frequencies of >6 Hz, I-only inhibition typically converts to excitation. We conclude that single whisker low-frequency stimulation can selectively block the spontaneous discharge of neurons in SI barrel field septa. The observation that this cell response is found most often in or at the edge of septa and at relatively long latencies supports the idea that I-only inhibition is mediated through cortical circuits. We propose that in these cells inhibition alone or a combination of inhibition and disfacilitation play a role in suppressing neuronal discharge occasioned by low frequency contact of the whiskers with the environment.
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INTRODUCTION |
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The cortical representation of
each whisker in the rodent somatic sensory (SI) cortex has been
referenced to clusters of neurons in layer IV (called barrels by
Woolsey and Van der Loos 1970) that respond to
deflection of whiskers on the contralateral face (Welker
1971
). Extracellular recordings in awake-alert (Fanselow and Nicolelis et al. 1999
; Fee et al. 1997
;
Nicolelis et al. 1995
; Simons et al.
1992
), awake-paralyzed (Simons 1978
),
and anesthetized (Armstrong-James and Fox 1987
;
Ito 1985
; Welker 1971
) rats show that
neurons in barrels typically respond to principal whisker deflection by
increasing their discharge rate 6-10 ms after the deflection.
GABAergic inhibition typically follows the excitatory discharge thereby
creating an "inhibitory trough" in the cumulative spike profile of
a poststimulus time histogram (Carvell and Simons 1988
;
Simons 1978
, 1985
). Deflection of
nonprincipal whiskers in the receptive field (RF) of cortical neurons,
the excitatory "surround" whiskers, also increases cortical
discharge rate, but at a longer latency and a lower magnitude than the
principal whisker (Armstrong-James and Fox 1987
). Using
a two whisker stimulation paradigm Simons and colleagues have shown
that surround whiskers can produce inhibitory interactions between
neurons in adjacent barrels and septa (Brumberg et al.
1999
; Simons 1985
). The response evoked by
principal whisker deflection is much reduced if an adjacent whisker is
deflected 2-50 ms before the principal whisker (maximum inhibition
~20 ms). These results raise the possibility that stimulation of
whiskers could directly inhibit the spontaneous discharge of cortical
neurons under some conditions.
Intracellular recordings in vivo from anesthetized rats
indicate that the initial response of barrel neurons to whisker
deflection is typically an excitatory postsynaptic potential (EPSP)
(Carvell and Simons 1988; Moore and Nelson
1998
; Zhu and Connors 1999
; Zhu and
Sakmann 1998
). EPSPs are followed by an inhibitory potential (IPSP); in fact EPSP-IPSP-EPSP sequences have been described in several
cortical areas, including rat barrel cortex and cat SI whisker cortex
(Hellweg et al. 1977
; Zhu and Connors
1999
; also see Kleinfeld and Delaney 1996
).
However, another sequence of events following whisker stimulation has
also been described. An in vivo study using whole cell patch recording
methods reported that 1 of 24 cortical neurons responded to a single
whisker exclusively with IPSPs (Moore and Nelson 1998
).
Stimulation of thalamocortical fibers in brain slice preparations have
also evoked solitary IPSPs in SI cortex (Agmon and Connors
1992
). Here we present evidence from extracellular
recordings in awake rats for suppression of spontaneous discharge
following whisker stimulation, without any preceding excitation.
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METHODS |
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All methods were approved by the University Animal Care Committee and were in accordance with NIH approved procedures.
Habituation to restraint
Male rats (n = 5) were handled every day for a
week and placed on a reduced diet. Rats were habituated to being
restrained by wrapping a towel around the animal and placing them in a
loosely fitting cloth bag. The rat in the bag was slid into a loose
fitting plastic tube where they were offered chocolate milk during the restraint from a lick tube. Restraint was kept as short as possible, typically 20-30 min of licking chocolate milk; rats gained 20-30 g in
a session. Once rats demonstrated that they would lie quietly and drink
chocolate milk, they were prepared for surgery. Rats were anesthetized
with pentobarbital sodium (Nembutal, 50 mg/kg) and a craniotomy was
made over SI cortex. Five small holes were made in the skull, three
over the cerebellum and two over rostral locations near the olfactory
bulbs. Holes in the skull were tapped and blunt tipped screws were
inserted. Using dental acrylic, a head post was fixed over the
cerebellum (Bermejo et al. 1996) and a chamber was
placed over the craniotomy. The chamber was sealed by a cap that could
be unscrewed to give daily access to the exposed dura. Once animals
recovered from the surgery, they were reacclimated to the restraint.
Animals were monitored during head post restraint to see if they showed
any signs of distress. Animals that frequently moved or made noises
when their head was immobilized were eliminated from this study.
Recording
Rats were awake, quiet, and restrained during recording (Fig. 1). They were fed chocolate milk during the recording session between epochs of whisker deflection. Two miniature screw-advance microdrives were mounted into grids fitted over the chambers. When screwed onto the grids, the microdrives independently advanced single tungsten wire electrodes (FHC) into the cortex. At the outset electrode tips were separated by roughly 1 mm. As the electrodes penetrated through the dura, neuronal responses could be heard on an audio monitor in response to manual stimulation of the whiskers. For each electrode one whisker was selected as the principal whisker based on the response magnitude.
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At the outset a period of spontaneous discharge was recorded for each neuron. The principal whisker for each electrode was deflected (~3 mm for 30 ms) at 1 of 8 stimulus frequencies (0.5, 1, 3, 6, 9, 12, 15, and 18 Hz) with an air puff stimulator constructed inhouse (James Long Company, Caroga Lake, NY). Data were collected for 50 s at all stimulus frequencies. Air puffs at each frequency in succession were directed from above the whisker pad. Whiskers were observed through an operation microscope throughout recording to ensure that only one whisker moved.
Electrodes were advanced in 75 µm steps. After cells in 3-5
depths had been sampled, lesions were placed at the bottom of each
electrode penetration (3 µA, for 10 s). To distinguish between the two tracks, one of the two electrodes was advanced by 300 µm, the other was retracted by 300 µm, and a second lesion
was made. The animal was killed with carbon dioxide, perfused with 0.1 M phosphate buffer, and the brain fixed with 4% paraformaldehyde. Once
the brain sank in 30% sucrose, the cortex was removed, flattened between slides, and cut into 50 µm thick sections tangential to the
cortical surface on a freezing microtome. Brains were stained for
cytochrome oxidase (Wong-Riley and Welt 1980)
and electrode tracks were reconstructed from serial sections. All data
included in this paper are from animals with electrode tracks
reconstructed and localized in the barrel field. In every case it was
possible to specify whether a recording site was in a barrel or septum.
Data acquisition and analysis
A head stage (NB Labs) was used to connect a multichannel connector to the two electrodes on the rat's head and to a multichannel neuronal spike data acquisition processor (Plexon, Dallas, TX). All waveforms from both electrodes were collected and saved for offline spike sorting. Units were rediscriminated offline using principal components and cluster cutting (Plexon). Stimulus evoked poststimulus time histograms and raster displays were constructed from the spike trains of the discriminated units.
Stimulus
Air puffs controlled for force and duration were used for stimulating the whiskers. The air puff is a supramaximal stimulus with a ramp-and-hold puff of air (~200 mm/s).
STIMULUS LATENCY. Time 0 for the stimulus onset was triggered by a solenoid opening on the air line several feet from the animal's face. Air travelling from the solenoid to the air outlet required 25 ms, and neuron response latency was calculated by subtracting 25 ms from the time to first spike for each trial.
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RESULTS |
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The data presented here focus on cells that decreased their spontaneous discharge in a stimulus-linked fashion. In each animal, anywhere from 6 to 10 recording sites were tested, and at each site, multiple units were discriminated from two electrodes. A total of 48 units were discriminated of which 10 responded to whisker stimulation with I-only inhibition. Autocorrelation functions for four units discriminated from four separate electrodes in two animals are shown in Fig. 2. The striking feature of these recordings is that whisker stimulation inhibits cell discharge, without any detectable excitatory discharge. In all recordings, histology showed that the electrode was on a barrel edge or clearly in a septal zone (Figs. 3A, 4A, and 5A). Except for a single recording site where stimulation of a large mystacial whisker (C2) inhibited the spontaneous discharge, the rest of neurons in the sample responded to small whiskers (nose hair, D6, and D7) that the animal typically does not move much with suppression of spontaneous activity.
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Stimulation of the C2 whisker at a frequency of 1 Hz inhibited the discharge of neurons recorded from electrode 2 (Fig. 3C, right PSTH) while the neuron on electrode 1 (Fig. 3C, left PSTH) was excited by C2 stimulation. Inhibition was restricted to the C2 whisker, as other whiskers (A2 and B1 are shown) did not evoke the same inhibition in the cortical cell.
In another animal, stimulation of the D6 whisker inhibited neurons on one electrode, while at the same time weakly exciting neurons on the other electrode (Fig. 4). Recording sites for the two electrodes were the edge of E7 whisker barrel and between C5, D6, and D7 barrels. One electrode had a receptive field of "F-row" whiskers that have no barrels associated with them, while the principal whisker for electrode 2 was the D6 whisker. At two recording sites, stimulation of the D6 whisker at 1 Hz inhibited spontaneous discharge of neurons.
Similarly, stimulation of the D7 whisker inhibited neurons at only one of the recording sites in another animal (not shown). At the other recording sites in the same animal, the D7 whisker evoked either no response or an excitatory discharge. Inhibition is not restricted to mystacial whisker stimulation. Nose hair stimulation can also inhibit and excite neurons in the barrel field (Fig. 5C).
Effect of stimulus frequency
The neurons just described respond differently at higher stimulus frequencies. Figures 6, 7, and 8 show a more complicated pattern of modulation that develops at higher stimulus frequencies. The same neuron shown in Fig. 3 (electrode 2) shows a modulated pattern of discharge that is double the stimulus frequency at 12 Hz (Fig. 6). Inhibition begins 15 ms poststimulus and lasts 125 ms poststimulus. At higher stimulus frequencies, one-half (5 of 10) of the neurons show a switch from an apparent inhibitory trough to an excitatory discharge following the stimulus (Fig. 7). In the other neurons, there is a mixed excitatory/inhibitory response to the stimulus, where the neuron is inhibited for a large sequence of trials, but apparently cannot maintain this response to whisker stimulation (Fig. 6 and 8).
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Recording site
All recording sites were in SI barrel cortex, either on the edge of a barrel or in the septum between barrels. In 4 of 5 animals in which whisker stimulation evoked an inhibitory discharge, the most superficial recording sites were in layer II, III, or IV. In the remaining animal, whisker stimulation evoked an inhibitory discharge only in the deepest recording site in layer V.
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DISCUSSION |
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The principal finding in this study was that whisker stimulation in the awake rat can block spontaneous discharge without evoking prior excitation. This suppression of discharge occurs at longer latencies than the fastest excitation produced at low stimulus frequencies. The cells showing I-only inhibition were found only in or at the border of septa.
Very early in the history of recording from single units in cat SI
cortex, Mountcastle (1957) reported that stimulation
with an air puff in the center of the receptive field evoked
excitation, but stimuli directed outside of a neuron's excitatory
receptive field inhibited spontaneous discharge in cortical cells. An
in vivo study carried out with intracellular methods in cat SI cortex (Hellweg et al. 1977
) showed that the most frequent
response to whisker stimulation was EPSPs followed by IPSPs. In the
center of the receptive field where inhibition was the strongest, IPSPs occurred after excitation. Outside the center of the receptive field,
however, IPSPs could precede EPSPs. Even further out in the receptive
field, inhibition could occur as the only response to whisker
stimulation. In this study no such gradation of inhibition has been
detected. Specific whiskers inhibited the spontaneous discharge of
neurons when other whiskers either evoked an excitatory discharge or
had no effect.
In the rat whisker cortex, surround whiskers typically evoke an excitatory discharge with little evidence of an initial inhibition. In this study, we made no attempt to determine whether the entire receptive field of a neuron was inhibitory, but clearly the same neuron whose spontaneous discharge was inhibited could respond with an excitatory discharge to a neighboring whisker. A neuron whose response was suppressed by whisker stimulation could even generate an excitatory discharge to the same whisker when it was stimulated at a higher stimulus frequency.
A number of previous studies of the rat vibrissal cortex have described
inhibition that follows excitation (Brumberg et al. 1996; Carvell and Simons 1988
; Kyriazi
and Simons 1993
; Kyriazi et al.
1996
; Simons 1985
; Simons and
Carvell 1989
), and in addition, there is some evidence for
I-only inhibition in cortical neurons (Hellweg et al.
1977
; Moore and Nelson 1998
). The
excitation-inhibition is very characteristic of the responses generated
by VPM to barrel thalamocortical inputs when activated by the principal
whisker. In the septa between layer IV barrels there is a dense input
from the POm nucleus rather than VPM (Koralek et al.
1988
; Lu and Lin 1993
), and septal cells often
respond equally strongly to more than one whisker
(Armstrong-James and Fox 1987
). The I-only inhibition has not been analyzed in the same detail, but its relatively long latency to onset is consistent with being generated by intracortical circuits rather than thalamocortical activation. An alternative explanation for both the postexcitatory inhibition and inhibition alone
might be that at least part of the suppression of cortical discharge
could be due to disfacilitation (the removal of drive onto the neuron)
and not due to active inhibition (Contreras et al. 1996
;
Cowan and Wilson 1994
). Arguments for this view are as
follows: 1) input resistance of cortical neurons is higher during long-lasting hyperpolarizations evoked spontaneously or by
thalamic stimulation and 2) GABAergic inhibitory mechanisms in cortex are relatively short compared with this inhibition. According
to this view, active inhibition has a role in suppressing neuronal
discharge, but this effect is short in duration, as short as the effect
of excitation in increasing neuronal discharge. More important to this
view, is the removal of all synaptic input, which increases the neurons
input resistance and suppresses the neurons discharge. This study is an
extracellular study and cannot distinguish between these possibilities.
Other studies
In this study the majority of neurons responded with excitation
followed by inhibition and had similar characteristics to those
reported previously by Simons, Carvell and colleagues (Brumberg et al. 1996; Simons 1985
). Evidence of
inhibition detected extracellularly can be seen 20 ms poststimulus and
can last 100-150 ms (also see Kleinfeld and Delaney
1996
). Inhibitory interactions in the rat vibrissal S1 cortex
have been studied with a dual whisker stimulation paradigm to look at
the effect of stimulating an adjacent whisker before, after, or during
principal whisker stimulation. These studies have shown that as the
number of stimulated adjacent whiskers increases, inhibition of
principal whisker evoked responses increases (Brumberg et al.
1996
). Adjacent whisker deflection within 10-20 ms suppresses
the response of a neuron to principal whisker deflection (Simons
1985
). One implication of these results is that whisker stimulation could have an inhibitory effect on neurons in barrel cortex. This study confirms that some septal neurons are directly inhibited by whisker stimulation. This study also suggests that the
inhibitory effect of low frequency whisker stimulation can be seen even
without dual whisker stimulation or the application of GABA agonists
and antagonists.
Methodological issues
During whisker stimulation the rat can move its whiskers. It is possible that the whisker stimulus dependent inhibition described here is related to movement of the whiskers. However, this possibility can be ruled out at least for some of recording sites, like the nose hair, because the rat cannot move his nose hair voluntarily. Waxing and waning of attention as a cause of whisker stimulus related suppression is harder to rule out.
Implication
In most layers of barrel cortex, 15-20% of the neurons are
GABAergic (Beaulieu 1993; Chmielowska et al.
1988
; Lin et al. 1985
). Layer IV is exceptional
in that it contains a higher percentage of GABAergic neurons with
estimates up to 50%. All neuronal elements in layer IV of mouse SI
barrel cortex receive inhibitory input from GABAergic neurons in layer
IV (Keller and White 1987
). Both inhibitory and
excitatory neurons receive thalamocortical excitatory inputs
(White 1979
, 1989
), making the initial response to
whisker deflection excitatory to all cell types, followed by a single synapse delay for the inhibition to take effect. Both recurrent excitatory and recurrent inhibitory connections within cortex likely
play an important role in the generation of each cortical neurons
response to whisker stimulation.
This study suggests that whisker stimulation can stop the firing of
spontaneously active septal neurons. Behaviorally, one interpretation
might be that when a rat's nostril, nose hair, or particular whiskers
are in contact with an object, input from other sensory inputs can be
blocked at the cortical level. This instruction could operate to
channel attention under behavioral conditions when a potential food
source is close to or in contact with the nose (Brecht et al.
1997). The modulation of spontaneous neuronal
discharge by inhibition might also help to synchronize the discharge of
neurons at a particular frequency.
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FOOTNOTES |
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Address for reprint requests: F. F. Ebner, Dept. of Psychology, 301 Wilson Hall, 111 21st Ave. South, Vanderbilt University, Nashville, TN 37240 (E-mail: ford.ebner{at}vanderbilt.edu).
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 6 January 2000; accepted in final form 19 May 2000.
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REFERENCES |
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