Download Basal Forebrain Cholinergic System Is Involved in Rapid Nerve

J Neurophysiol 91: 424 – 437, 2004.
First published September 24, 2003; 10.1152/jn.00489.2003.
Basal Forebrain Cholinergic System Is Involved in Rapid Nerve Growth
Factor (NGF)-Induced Plasticity in the Barrel Cortex of Adult Rats
Neal Prakash,1 Susana Cohen-Cory,1 Silke Penschuck,1 and Ron D. Frostig1–3
Departments of 1Neurobiology and Behavior, 2Biomedical Engineering, and the 3Center for the Neurobiology of Learning
and Memory, University of California, Irvine, California, 92697-4550
Submitted 21 May 2003; accepted in final form 15 September 2003
Prakash, Neal, Susana Cohen-Cory, Silke Penschuck, and Ron D.
Frostig. Basal forebrain cholingergic system is involved in rapid
nerve growth factor (NGF)-induced plasticity in the barrel cortex of
adult rats. J Neurophysiol 91: 424 – 437, 2004. First published September 24, 2003; 10.1152/jn.00489.2003. We have previously reported that topical application of nerve growth factor (NGF) to the
barrel cortex of an adult rat rapidly augmented a whisker functional
representation (WFR) by increasing its area and height within minutes
after NGF application. In addition, we found that TrkA, the highaffinity NGF receptor, was only found on fibers projecting into the
barrel cortex. Here we use a combination of techniques including
chronic intrinsic signal optical imaging, neuronal fiber tracking and
immunohistological techniques, to test the hypothesis that NGF-induced rapid cortical plasticity is mediated by the cortical projections
of the basal forebrain cholinergic system (BFCS). Our studies localize
the source of the cells in the BFCS that project to a single WFR and
also demonstrate that TrkA-immunoreactive fibers in the cortex are
also cholinergic and likely arise from the BFCS. In addition, by
selectively lesioning the BFCS cortical fibers with the immunotoxin
192 IgG-saporin, we show that NGF-induced WFR-cortical plasticity
is eliminated. These results, taken together with our previously reported imaging results that demonstrated that agonists of the cholinergic system (particularly nicotine) showed transient NGF-like augmentations of a WFR, implicate the BFCS cortical projections as
necessary for NGF’s rapid plasticity in the adult rat somatosensory
cortex.
Previously we reported that neurotrophins, such as nerve
growth factor (NGF) and brain-derived neurotrophic factor
(BDNF), can rapidly induce large-scale plasticity in the adult
somatosensory cortex (Prakash et al. 1996a,b). For example,
within minutes after topical application to the cortex, NGF
caused a transient, large-scale expansion in the area of a
whisker functional representation (WFR) coupled with an increase in the height of the WFR. The current study attempts to
describe potential underlying mechanisms that mediate this
NGF-plasticity. One possibility may be that NGF is acting
directly on cortical neurons to induce such plasticity. However,
most evidence indicates that in the adult rat, NGF receptors are
not expressed by cortical neurons but rather in neurons that
project to the cortex (Merlio et al. 1992) and on projection
fibers to the somatosensory cortex (Holtzman et al. 1995;
Prakash et al. 1996b). This evidence thus suggests that NGFinduced adult cortical plasticity should involve neuronal projections into the cortex that are presynaptically responsive to
NGF. Here we present evidence that indicates that the basal
forebrain cholinergic system (BFCS) is the source of these
cortical projection fibers and thus implicates the BFCS in the
NGF-induced cortical plasticity.
The basal forebrain (BF) in the rat, known as the magnocellular basal forebrain nuclei, includes cells located in the
nucleus basalis of Meynert and the septal nucleus of the diagonal band of Broca (Fibiger 1982; Sofroniew et al. 1982; Wenk
et al. 1980). Cholinergic cells within this system project to the
entire rat cortex (Lehmann et al. 1980) in a loose topographically organized manner (Jimenez-Capdeville et al. 1997; Lamour et al. 1982) and constitute the only known cholinergic
projection to the adult cortex (Mesulam et al. 1983). After
release, acetylcholine (ACh) can influence target cells in the
cortex through two broad classes of cholinergic receptors:
muscarinic and nicotinic receptors.
It has been demonstrated that NGF is synthesized in the
cortex by the target cells of BF projections in the cortex and
that it binds to receptors located on the BF nerve terminals and
is retrogradely transported to cell bodies of BF neurons, where
it regulates their survival and function as predicted by the
neurotrophic hypothesis (Cuello et al. 1992; Hefti et al. 1989).
While extensive evidence exists for the neurotrophic hypothesis in the developing brain, evidence also exists to support the
notion that NGF continues to play a maintenance function for
the BFCS after the brain matures. Exogenous application of
NGF can rescue axotomized BF cholinergic neurons, reverse
the decline in cortical function, and restore memory (Dekker et
al. 1992; Gage et al. 1988; Jacobs et al. 1994; Koliatsos et al.
1990; Kromer 1987; Will and Hefti 1985; Williams et al.
1986). More direct experimental proof for the role of endogenous cortical NGF in the maintenance of the BFCS (Minger
and Davies 1992a,b) was obtained when local injections of
anti-NGF antibodies were delivered into the cortex of the adult
rat (Gutierrez et al. 1997). These rats demonstrated a lack of
ACh release in the cortex, a disruption of connectivity between
cortex and BF, and a disruption of learning and memory. The
importance of endogenous cortical NGF was also demonstrated
by the use of mutant mice (Chen et al. 1997). Heterozygous
mutant mice for the NGF gene showing a reduced level of both
NGF-mRNA and protein within the cortex had significant
learning and memory problems that were accompanied by a
loss and shrinkage of BF cells. Infusion of NGF in these adult
mutants abolished the memory deficits and corrected the def-
Address for reprint requests and other correspondence: R. D. Frostig, Dept.
of Neurobiology and Behavior, University of California, Irvine CA, 926974550 (E-mail: [email protected]).
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.
INTRODUCTION
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0022-3077/04 $5.00 Copyright © 2004 The American Physiological Society
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MECHANISMS UNDERLYING NGF CORTICAL PLASTICITY
icits in both area and projection density of BF cells to the
cortex.
It is clear that NGF plays a role in maintaining the BF
cholinergic projections to the cortex. Two key findings have
also suggested the possibility that NGF can also induce immediate release of ACh. Knipper and collaborators obtained in
vitro evidence for NGF-induced rapid release of ACh in the
cortex (Knipper et al. 1994a,b). The authors demonstrated such
effects in synaptosomes prepared from the rat hippocampus
that included BF projection fibers. They showed enhanced
ACh release from these synaptosomes starting within 1 min
after NGF application. Anti-NGF antibodies blocked the NGFinduced rapid release of ACh. The rapid induction of ACh
release cannot be readily explained by NGF’s known role of
regulating the survival and function of BF cholinergic system
as this role requires a longer window of time (hours to days) for
these effects. In addition, the rapid release of ACh supports the
existence of NGF receptors on the terminals BF projection
fibers. Rapid induction of ACh release by NGF was also
demonstrated in synaptosomes prepared from the visual cortex
of rats and further demonstrated that such induction of ACh
release involves the activation of the high-affinity TrkA receptor and the p75 low-affinity neurotrophin receptor (p75) (Sala
et al. 1998). Further, we obtained in vivo evidence for NGF’s
ability to rapidly increase (within minutes after application) the
height and areal extent of whisker evoked activity in the
somatosensory cortex (Prakash et al. 1996b). Our previous
histological results suggested that NGF’s site of action is not
directly on the neurons within the somatosensory cortex but
rather on projections originating outside it. Given that BFCS
projection neurons are the only type of neurons with both
projections to the cortex and NGF receptors (Holtzman et al.
1995), our findings (Prakash et al. 1996b) suggested an in vivo
link for NGF’s rapid in vitro effects that may be mediated by
the BFCS.
One possible mechanism for NGF’s induced plasticity is that
under behavioral conditions when cortical representations need
to be rapidly augmented by increasing cortical excitability and
by recruiting more neurons, cortical neurons release NGF in an
activity-dependent manner in addition to the basal release of
NGF needed for the survival and maintenance of the BF. The
released NGF binds to its receptors (TrkA and p75) located on
the BFCS projection fibers in the cortex. The binding of NGF
then enhances the release of ACh from BFCS projections
within the cortex. The release of ACh, in turn, further enhances
cortical activity through ACh receptors and potentially glutamate release enhancement by ACh. These actions, in turn, lead
to an increase in the excitability of neurons within a WFR and
the recruitment of additional neurons to the WFR, leading an
areal expansion and increased height of the WFR as observed
with the imaging results (Prakash et al. 1996b). Elsewhere we
reported, in support of this hypothesis, that activation of muscarinic or nicotinic receptors in the somatosensory cortex results in a WFR plasticity that is similar to the NGF-induced
plasticity (Penschuck et al. 2002). Here we present data that
tested three other critical aspects of our hypothesis: we demonstrate that projections from BFCS reach the level of a single
WFR in barrel cortex; we demonstrate that TrkA-immunoreactive fibers present in the somatosensory cortex are also
cholinergic fibers that likely arise from the BFCS; and we show
that specifically lesioning the BFCS fibers in the barrel cortex
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results in a loss of the ability of NGF to rapidly augment a
WFR. Some of these data have been published previously in
abstract form (Prakash et al. 2000a).
METHODS
Imaging
We have followed a procedure described in Prakash et al. (1996b)
and is described briefly here. Adult male Sprague-Dawley rats (300 –
550 g) were initially anesthetized with pentobarbital (50 mg/kg ip).
Throughout the rest of the experiment, pentobarbital was injected as
needed to maintain the rat in a consistent anesthetic state such that the
respiration rate was 1.1 ⫾ 0.2 (range) breaths/s, rectal temperature
was 37.0 ⫾ 0.5°C, ear color was pinkish-pale, and the corneal reflex
in response to a saline drop was weak to mild. Lidocaine (0.1 ml) was
injected under the scalp, and 5 min later a single incision was made
with a scalpel roughly along the midline of the scalp. Fascia and
muscle overlying the parietal cortex were cleared, and the bone was
cleaned and dried. Next, the skull overlying the somatosensory cortex
was removed. However, unlike the previous protocol (Prakash et al.
1996b) in which the dura and arachnoid mater were removed (durectomy), in this set of experiments, the dura remained in place but small
slits or holes were made in it (durotomy) using a 30-gauge needle.
This change in protocol was instituted to decrease the probability that
rats would have significant brain herniation. A petroleum-jelly well
was then built around the partially exposed cortex, filled with artificial
cerebrospinal fluid [ACSF, containing (in g) 7.250 NaCl, 0.370 KCl,
0.170 KH2PO4, 0.277 CaCl2, 0.290 MgSO4, 2.160 NaHCO3, and
1.800 glucose (all per 1 l H20), pH 7.30, 37°C], and sealed with a
coverslip.
A 12-bit slow-scan CCD camera (Photometrics, Tucson, AZ) imaged a 6.8 ⫻ 5.1 mm (192 ⫻ 144 pixels; 35.5-␮m pixel width) region
of the cortex that was illuminated with a stabilized 100-W tungstenhalogen light source (Zeiss Instruments) passed through an optical
filter (630 nm band-pass 30 nm; Omega Optics, Brattleboro, VT). To
reduce potential surface artifacts induced by pulsations of the brain
and vessels, the camera was focused 300 ␮m below the surface of the
cortex. The light reflectance was measured for each pixel (9 consecutive frames, 500 ms each) before, during, and after stimulation of the
contralateral whisker(s). Whisker stimulation was given with a computer-controlled mechanical stimulator (Bakin Systems II, Irvine, CA)
at 5 Hz for 1 s. The interstimulus interval between trials was 16 s.
Data files were created by the summation of 64 trials and thus had
duration of ⬃20 min. The right C2 whisker was deflected 2° rostrocaudally (0.5 mm at 1.5 cm from the snout) with an average velocity
of 30°/s. Eight imaging data files were collected per rat. The first four
data files served as baseline reference. Additionally as an internal
control to determine the affects of change the ACSF in the petroleumjelly well, the ACSF was replaced between data files 2 and 3. No
significant difference in the measured parameters was ever noted after
this ACSF change. After the fourth data file, the ACSF was replaced
with recombinant human NGF dissolved in ACSF (15 ␮g in 500 ␮l).
Quantitative analysis of a whisker functional representation
Analysis methods have been described in detail elsewhere (ChenBee et al. 1996; Masino and Frostig 1996; Masino et al. 1993; Prakash
et al. 1996b, 2000b). Briefly, data files were created from the summation of 64 imaging trials. Ratio values for a data file were computer-calculated for each pixel by dividing the reflectance values of
the images 0.5–1.5 s poststimulation by the images 1.0 – 0.0 s before
stimulation. Before analysis, a Gaussian filter (half-width 5) was
applied to the raw ratio values to filter out high-frequency noise. The
peak location of a WFR is defined as the pixel with the most negative
ratio value, and this ratio value minus the median ratio value is the
peak height of a WFR, which indicates the maximal magnitude of
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N. PRAKASH, S. COHEN-CORY, S. PENSCHUCK, AND R. D. FROSTIG
evoked activity. The area at half-height of a WFR is the area bounded
by the threshold that contains pixels 50% of the peak height value.
Relationship between imaging and underlying neuronal
activity
We focused our analysis on reflectance changes of 630-nm light
occurring between 0.5 and 1.5 s poststimulus onset, an epoch of
activity predominantly consisting of localized oxygen consumption in
the capillary bed serving activated neurons (Ances et al. 2001; Frostig
et al. 1990; Kim et al. 2000; Malonek and Grinvald 1996; Malonek et
al. 1997; Thompson et al. 2003) as the area of imaged activity
generated from this epoch has been previously found to be well
correlated with the underlying area of evoked neuronal activity (BrettGreen et al. 2001; Frostig et al. 1990; Masino 2003; Polley et al.
1999). We also have repeatedly found a strong correspondence between the spatial distribution of intrinsic signals and the underlying
single-unit responses such that the location of the strongest single-unit
response for each rat coincided with the location of that rat’s strongest
intrinsic signal response; evoked single-unit responses were present at
all locations containing evoked intrinsic signal responses; and both
responses decayed similarly with increasing distance from the location of peak activity (Brett-Green et al. 2001; Masino 2003; Polley et
al. 1999). Furthermore, postimaging single-unit recordings demonstrated that plasticity of a WFR (both in its areal extent and height)
was identical to the one obtained with intrinsic signal optical imaging
(Polley et al. 1999). Thus for both the normal functional organization
of cortex and its plasticity, intrinsic signal optical imaging that is
based on localized oxygen consumption has been shown to be a
reliable indicator of underlying physiological neuronal activity patterns and their plasticity.
Retrograde labeling of basal forebrain cells
projecting to barrel cortex
Fifteen male Sprague-Dawley adult rats (300 – 600 g) were imaged,
and the exact location of the peak of activity for the C2 WFR was
determined using stereotactic methods. The location of Bregma was
defined as the origin, and C2 WFR location was measured in relation
to the origin using its peak activity as obtained by imaging through the
thinned skull (⬃150 ␮m thickness). The average location of C2 was
useful in other experiments, such as the 192-IgG-saporin injections
described in the following text, and in localizing the C2 barrel in
coronal sections. A small burr hole was drilled through the cortex over
the peak activity of the WFR. Approximately 1 ␮l Fast-DiI oil
(1,1⬘-dilinoleyl-3,3,3⬘,3⬘-tetramethylindocarbocyanine perchlorate,
Molecular Probes, Eugene, OR) dissolved in dimethylsulfoxide was
then pressure injected (PicoSpritzer II, General Valve, Brookshire,
TX) through a glass pipette that was lowered 500 ␮m below and
perpendicular to the pial surface. Fast-DiI is a fluorescent molecule
that travels along lipid membranes, thus staining axons (anterogradely
and retrogradely) and cell bodies (predominantly retrogradely). After
waiting 3 min, the pipette was slowly retracted, and the burr hole was
sealed with sterile bone wax. The scalp incision was closed with
wound clips, and prophylactic antibiotics were administered (Panalog
(nystatin, neomycin sulfate, thiostrepton, triamcinolone acetonide) on
the eyes, Terramycin over the wound, and Ampicillin subcutaneously
in the back). Rats were survived for 6 –10 days and then given an
overdose of sodium pentobarbital (Nembutal, 100 mg). When completely areflexive, rats were perfused transcardially with 0.1 M PBS at
4°C (⬃150 ml) followed by fixation with a solution containing 4%
paraformaldehyde and 0.25% gluteraldehyde in 0.1 M Sorensen’s
buffer (⬃150 ml). The brain was removed and postfixed in the same
solution overnight, followed by immersion for 2–3 days in 30%
sucrose dissolved in 0.1 M PB. On sinking, the brain was cut coronally on a cryostat at –20°C; each section was thaw-mounted serially
directly onto gelatin-coated slides. As DiI is prone to dissolution after
J Neurophysiol • VOL
sectioning, sections were viewed either dry or with 0.1 M PBS and a
coverslip. The locations of DiI-positive cell bodies throughout the
basal forebrain were noted and photographed using a Rhodamine filter
on a fluorescent microscope (Nikon).
Techniques to detect cholinergic neurons and fibers
Cholinergic neurons in the nervous system produce the neurotransmitter acetylcholine by the enzyme choline acetyl transferase (ChAT).
ACh is then transported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). After acetylcholine is released at the
synapse, it is rapidly degraded by acetylcholine esterase (AChE).
Based on these three enzymes, different histochemical methods have
been developed to visualize cholinergic neurons. AChE visualization
techniques are the oldest and best-characterized staining techniques
for cholinergic neurons and fibers. These techniques are the mostsensitive visualization techniques but are not specific enough because
norepinephrinergic and dopaminergic neurons may also stain positive
for AChE (Albanese and Butcher 1979; Henderson 1989; Tago et al.
1986). Immunohistochemical techniques for staining neurons and
fibers for either ChAT of VAChT have proven to be more specific for
cholinergic neurons but are generally less sensitive at elucidating
cholinergic fibers than AChE staining (Armstrong et al. 1983; Eckenstein and Sofroniew 1983; Holler et al. 1996; Mizukawa et al. 1986;
Satoh et al. 1983; Weihe et al. 1996). Therefore depending on whether
we required sensitivity or specificity, all three staining methods were
chosen for different experiments described in the following text.
Co-localizing TrkA and ChAT
We required maximal specificity to demonstrate that cholinergic
fibers were also expressing TrkA. Therefore ChAT was chosen for
these experiments. Initial studies were conducted to optimize the
single staining protocols for TrkA and ChAT, and in few rats, double
staining with VAChT and TrkA was also performed. A rat was given
an overdose of pentobarbital and then perfused transcardially with
4°C PBS (0.1 M PBS, ⬃150 ml) and fixed with 4% paraformaldehyde
(⬃150 ml). The brain was removed and placed in the same fixative
overnight, then transferred to 30% sucrose in 0.1M PB until it sank
(⬃3 days). The brains were frozen (⫺20°C) and sectioned to 35-␮m
slices. For double staining, 20- to 35-␮m slices were incubated
simultaneously with goat anti-ChAT antibody (Chemicon International, Temecula, CA) and rabbit anti-TrkA antibody (Holtzman et al.
1995) in 5% donkey serum overnight at 4°C. After rinse in 0.1 M
Tris-buffered saline (TBS), sections were incubated for 1 h at room
temperature in darkness in Cy2-conjugated donkey anti-rabbit antibody and Cy3-conjugated donkey anti-goat antibody (both from Jackson ImmunoResearch Labs). Four controls were used that had identical treatment as the other slices except for the omission of one of the
following antibodies: anti-ChAT, anti-TrkA, anti-rabbit, or anti-goat.
After rinse in 0.1 M TB, sections were mounted on gelatin-coated
slides and dried for ⬃15 min. Sections were treated with Prolong
mounting media (Molecular Probes) and coverslipped and stored in
the dark at 4°C. One to 2 days later sections were visualized and
imaged under a fluorescent microscope (Nikon or Olympus) with a
FITC (for Cy2) or rhodamine (for Cy3) filter.
Cholinergic depletion of a cortical whisker functional
representation
IgG-saporin (192) is a cytotoxin that was developed by R.G. Wiley
and colleagues and has been demonstrated to be extremely effective
and specific at eliminating cholinergic basal forebrain cells (Book et
al. 1992, 1994; Sachdev et al. 1998; Wenk 1997; Wiley 1992; Wiley
et al. 1991) (Baskerville et al. 1997; Zhu and Waite 1998). IgGsaporin (192) works by binding to p75 receptors on cholinergic fibers,
where it is internalized and transported back to the nucleus. Saporin
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MECHANISMS UNDERLYING NGF CORTICAL PLASTICITY
then binds to ribosomes and irreversibly blocks protein synthesis,
which leads to cell death. One effective method of depleting the barrel
cortex of cholinergic inputs is by ventricular injection of 192 IgGsaporin (Baskerville et al. 1997; Zhu and Waite 1998; reviewed by
Schliebs et al. 1996; Wiley 1996). However, even more precise,
localized depletions of cholinergic inputs are possible by injection of
a small amount of 192 IgG-saporin directly into the cortex (Bucci et
al. 1998; Sachdev et al. 1998). Data obtained from such localized
injections is not confounded by interpretations related to possible
secondary effects from the complete cholinergic depletion of a hemisphere(s) and parts of the cerebellum, as obtained with intracerebroventricular injections (Wiley et al. 1991). To test the hypothesis that
cholinergic fibers are involved in NGF’s induced plasticity of a WFR,
we locally depleted cholinergic fibers within the C2-WFR, using a
similar protocol as Sachdev and collaborators who were also using
192 IgG-saporin to test cortical plasticity in the adult barrel cortex
(Sachdev et al. 1998). These authors reported that local injection of
192 IgG-saporin into the barrel cortex results in a loss of the normal
cortical plasticity that occurs between two-whiskers when all the other
whiskers are trimmed from a rat’s snout (Sachdev et al. 1998).
Combining chronic imaging with cholinergic depletion
Thirty rats were used in this study, 20 experimental and 10 controls.
Of the 20 experimental rats, 8 were successful for all 3 phases
described in the following text. Additionally 3 of the remaining 12
experimental rats were successful in the first two phases but had only
negligible AChE fiber depletion (⬎75% remaining) in the C2-barrel
column, as evident in phase 3. For the control rats, 6 of 10 were
successful for all 3 phases.
The right C2
WFR was imaged through a thinned skull in pentobarbital-anesthetized adult male Sprague-Dawley rats (300 –550 g). To avoid direct
damage to the C2 WFR, a burr hole in the skull was drilled between
1.5 and 2.0 mm just medial of the peak of activity of the C2 WFR.
Eight nanograms 192 IgG-saporin (Chemicon) dissolved in 2 ␮l 0.1 M
PBS with 0.05% sodium azide (or only 2 ␮l of PBS and 0.05%
sodium azide for controls) was pressure-injected (Picospritzer II,
General Valve) through a glass pipette 1,500 ␮m below the pial
surface. Similar to (Sachdev et al. 1998); this depth allowed for good
penetration and diffusion of the toxin throughout the cortical tissue.
PHASE 1—LOCALIZATION OF WFRS AND INJECTION.
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Also similar to Sachdev et al. (1998), the distance of 1.5–2.0 mm from
the C2 WFR was found to be close to the maximal distance for
inducing an effective lesion in C2 in this dosage range. This distance
also prevented direct damage to the C2 WFR from the pipette.
Additional care was taken to avoid traumatizing major cortical vasculature with the pipette. Once injected, the pipette was slowly retracted, and the burr hole was sealed with sterile bone wax. The scalp
incision was closed with wound clips, and prophylactic antibiotics
were administered (Panalog on the eyes, Terramycin over the wound,
and Ampicillin subcutaneously). Rats recovered from surgery overnight in a cage resting on a heated blanket, and then were returned to
individual cages in the vivarium for 21 days with free access to food
and water on a 12-h light, 12-h dark cycle.
PHASE 2—NGF-EFFECTS ON DEPLETED AND CONTROL CORTEX.
Similar to Sachdev et al. (1998), 21 days was chosen as the interval
between injection and second imaging of the C2 WFR; this interval
allowed for a full recovery from the surgery. Twenty-one days after
the first imaging session/injection, rats were briefly observed for gross
behavioral changes and then weighed. All rats gained between 5 and
100 g over the 21 days, and no gross behavioral changes were
observed. Rats were then anesthetized with intraperitoneal pentobarbital and local lidocaine and placed in the stereotactic apparatus. The
remaining wound clips were removed, and the incision site re-opened.
The “cortical window” in the skull that from the previous imaging
session was frequently revascularized and thus required cleaning and
cauterizing. Once cleaned, the cortical window was resected using a
dental drill and forceps. To allow access of NGF into the cortical
parenchyma, in all rats, three to four small slits (⬃100 ⫻ 1,500 ␮m
each; Fig. 8) were made in the dura and arachnoid mater using a
30-gauge needle. The slits were made in locations that surrounded and
touched the borders of the previously identified location of the C2
WFR and that avoided tearing the dural vessels. A petroleum-jelly
well was then built on the bone surrounding the cortical window and
filled with ACSF and topped with a coverslip. The imaging protocol
as described in the preceding text and previously (Prakash et al.
1996b) was then used to assess the C2 WFR before and after application of NGF.
PHASE 3—QUANTIFYING THE CHOLINERGIC DEPLETION. At the end
of the imaging session, rats were perfused transcardially with 4°C 0.1
M PBS (⬃150 ml) followed by fixation with a solution of 4%
paraformaldehyde, 0.5% gluteraldehyde, and 0.2% picric acid in 0.1
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N. PRAKASH, S. COHEN-CORY, S. PENSCHUCK, AND R. D. FROSTIG
M Sorensen’s buffer (⬃150 ml). The brain was removed and placed
in 30% sucrose in 0.1 M PB until it sank. (Approximately half the
brains were prepared for tangential cutting: the cortex was dissected
from the rest of the brain and flattened to 2.0 mm between 2 slides and
then the slides and brain were placed in 30% sucrose). The brains
were then frozen to –20°C, and serial sections were cut coronally or
tangentially at 40-␮m thickness on a cryostat (Zeiss). Alternating
sections were stained for cytochrome oxidase (CO) (Wong-Riley
1979) to visual barrels and layer IV. The other alternating sections
were stained for AChE, using Tago’s modifications (Tago et al. 1986).
Additionally, slices were obtained from several rats and stained for
recombinant human NGF-immunoreactivity to ascertain the penetration of NGF through dural tears. This protocol was similar to as
previously described (Prakash et al. 1996b). Sections were mounted
onto gelatin-coated slides and allowed to dry, then covered with
mounting media followed by coverslips. Sections were analyzed and
imaged under a microscope (Olympus), and the location of the left and
right C2 barrels and their relations to the vascular patterns were
ascertained from the CO sections. The vascular patterns from the
adjacent AChE section were then compared with CO section vascular
patterns and used to determine the location of the C2-barrels in that
section. Photomicrographs (⫻20 or ⫻40) were then taken in each
C2-barrel and in layer I/II directly above the barrel. Photomicrographs
were digitized, and in PhotoShop 5.0 (Adobe, San-Jose, CA), a
100-␮m line was then drawn through the barrel in layer IV and above
the barrel in layer I/II. The line was drawn perpendicular to the pial
surface for coronal sections or horizontal for tangential sections.
“Fiber crossings” were calculated by adding up the number of intersections the two 100-␮m lines made with AChE-positive fibers. AChE
fiber depletion was defined as {1 ⫺ [(total fiber crossings on injected
side)/(total fiber crossings on control side)] ⫻ 100%}. Although there
were no obvious differences across the different lamina, we quantified
the fiber depletion in layers IV and layer I/II as these layers are
presumably the primary loci relevant to NGF’s effects. However,
there were differences in the fiber depletion in the C2 barrel across
different rats. This was most likely from the 1.5–2.0 mm variability in
injection site distance from the C2 barrel, which was introduced to
avoid damaging major blood vessels (see Phase 1 in the preceding
text). However, this variability across rats proved to be advantageous
because it allowed for a correlation of NGF’s induced plasticity with
the fiber depletion ratio (Fig. 4).
RESULTS
Regional topography of basal forebrain projection to a WFR
To determine whether the general pattern of BFCS projection cells to the somatosensory cortex holds even for a single
WFR, we injected Fast-DiI, a retrograde tracer, into the cortical
C2-whisker functional representation that resulted in transport
of tracer to several areas of the brain, such as the thalamus,
zona incerta, and basal forebrain, in similar patterns as we
reported in mice (Prakash et al. 2000b). Previous studies have
suggested that the somatosensory cortex of the rat receives
basal forebrain projections predominantly from the nucleus
basalis of Meynert [nBM; which consists of the ventral part of
the globus pallidus (vGP), the substantia innominata (S.I.),
some cells in the internal capsule, and a few cells scattered in
other nuclei (Bigl et al. 1982; McKinney et al. 1983)]. Our
primary interest in this study was to determine the precise
regions of the basal forebrain that were labeled. First we
wanted to verify the identities of the basal forebrain nuclei that
project to the adult rat barrel cortex. Second, we wanted to
determine whether an individual WFR had a more specific
basal forebrain projection locus than reported for the rest of the
J Neurophysiol • VOL
somatosensory cortex (Bigl et al. 1982; Kiss and Patel 1992;
McKinney et al. 1983). Consistent with these previous reports,
which examined the innervation of the entire somatosensory
cortex, we found that the C2-WFR was innervated by a scattered group of large cells throughout the nBM (Fig. 2). Other
cells were found scattered throughout the basal forebrain but
never in as high a concentration as the nBM. Overall, our
results suggest that the pattern of projections to a single WFR
is not more localized than the general pattern of BF cells that
project to the sensory cortex. These results were helpful for the
interpretation of the intracortical 192 IgG-Saporin injections
described in the following text.
Cytochemistry of basal forebrain projection cells and TrkA
cortical fibers
The basal forebrain contains at least two major classes of
neurons that project fibers to the cortex: parvalbumin-immunoreactive (-IR; GABAergic) and ChAT-IR (cholinergic). The
cholinergic fibers are the best candidates for also expressing
TrkA because previous work demonstrated that most of the
abundant ChAT-IR cell bodies in the basal forebrain are also
p75-IR/TrkA-IR (Sobreviela et al. 1994), whereas GABAergic
cells in the basal forebrain are p75-IR negative (Kiss et al.
1993). Although there is no question that cholinergic basal
forebrain cells also express TrkA, there was no direct evidence
that cholinergic projection fibers in the cortex co-express
ChAT and TrkA.
To assess whether TrkA-IR fibers observed in the cortex are
also cholinergic, co-localization studies with TrkA-IR and
ChAT-IR were performed. We found that TrkA fibers were
less abundant than ChAT fibers (⬃1:4 ratio), but that all
TrkA-IR fibers in the barrel cortex were also ChAT-IR (Fig. 1).
Furthermore, the TrkA fibers were sparsely distributed, but
present in all cortical layers (I–VI). To further verify this
finding, double-labeling studies with TrkA-IR and the vesicular acetylcholine transporter (VAChT)-IR were performed (not
shown). These experiments demonstrated similar types of
staining patterns as the ones presented in Fig. 1. In summary,
we found TrkA-IR only on fibers in the adult rat barrel cortex,
and all these fibers also were cholinergic (either ChAT-IR or
VAChT-IR).
Necessity of the bfcs for the expression of ngf’s rapid
plasticity
Confirming our observation that TrkA expression is found
exclusively in projection fibers into barrel cortex and that all
TrkA-IR fibers are cholinergic, we next analyzed the results of
depletion of the BFCS fibers in the barrel cortex (Fig. 2). This
depletion was accomplished by injecting 192 IgG-saporin into
the cortex near the C2-WFR and then re-imaging of the same
C2-WFR 3 wk later. Because of our histological studies using
Fast-DiI, we knew that the pattern of BF projection cells to a
single WFR is similar to the general patterns of BF projection
cells to the somatosensory cortex. Thus imaging-based localized injections of 192 IgG-saporin to the somatosensory cortex
near a single WFR would result in confined, yet representative
and thus interpretable, pattern of lesions in the BF. In these rats
the depletion was between 35 and 95%. A control group had
saline injections into the cortex near the C2-WFR, which had
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429
FIG. 1. TrkA-immunoreactive (IR) and choline acetyl
transferase-IR (ChAT-IR) co-localize in nucleus basalis of
Meynert cells and in cortical fibers. Thin sections of a rat
brain were double stained with specific antibodies to ChAT
and TrkA. Top: layer I of the barrel cortex. ChAT-IR fibers
are present near the pial surface of the cortex (top right). Of
these fibers, a few are also immunopositive for TrkA (top
middle), as shown by the overlaid image (yellow; see ).
Middle: TrkA-IR fibers found in layer V of the barrel
cortex. While ChAT and TrkA immunoreactivities are localized to individual fibers (), their localized distribution
within the fiber is not necessarily overlapping/restricted to
the same area. Bottom: immunostaining of a region of the
substantia innominata in adult rat nucleus basalis of Meynert (nBM; coronal section) shows large ChAT-IR cells that
are also immunopositive to TrkA. These images are typical
of all rats examined, and similar results were obtained using
an antibody to vesicular acetylcholine transporter.
no detectable AChE fiber depletion. Additionally, a third group
of rats had injection of 192 IgG-saporin that was subsequently
found to have minimal AChE fiber depletion at the C2-WFR
(between 0 and 25%). Both the control group (n ⫽ 6) and the
group with an AChE fiber depletion between 0 and 25% (n ⫽
3; Fig. 4) showed increases in both the area at half-height and
the peak height after NGF application that were not statistically
significantly different from each other (P ⬎ 0.05, t-test, n ⫽ 9).
We thus pooled the control group and the group with AChE
fiber depletion between 0 and 25% group together as the
“non-depleted group” (n ⫽ 9).
To determine if depleting the cortex of the BFCS affected a
WFR 21 days after 192 IgG-saporin injection, we compared
the area and strength of the C2-WFRs before application of
NGF in the depleted and non-depleted groups. There was no
difference between the C2-WFR of depleted and non-depleted
groups for either the area at half-height [1.38 ⫾ 0.16 vs. 1.25 ⫾
0.13 (SE) mm2, P ⬎ 0.05, t-test, n ⫽ 17] or the peak height
(2.64 ⫻ 10⫺4 ⫾ 0.25 ⫻ 10⫺4 vs. 2.39 ⫻ 10⫺4 ⫾ 0.46 ⫻ 10⫺4,
P ⬎ 0.05, t-test, n ⫽ 17). Therefore injection of 192 IgGsaporin locally into the cortex did not have a significant effect
on a WFR when compared across rats (Fig. 6, also compare
gray bars in Fig. 7).
To determine whether topical application of NGF-induced
WFR plasticity in a BFCS-depleted cortex, we examined the
area and strength of the C2-WFR before and after NGF application. In the depleted group, the C2-WFR did not change
significantly after topical application of NGF, for either area at
half-height (1.38 ⫾ 0.16 vs. 1.33 ⫾ 0.13 mm2, P ⬎ 0.05,
paired t-test, n ⫽ 8) or the peak height (2.64 ⫻ 10⫺4 ⫾ 0.25 ⫻
10⫺4 vs. 2.61 ⫻ 10⫺4 ⫾ 0.30 ⫻ 10⫺4, P ⬎ 0.05, paired t-test,
J Neurophysiol • VOL
n ⫽ 8). In contrast, in the non-depleted group both the area at
half-height (1.25 ⫾ 0.13 vs. 1.59 ⫾ 0.14 mm2, P ⬍ 0.05,
paired t-test, n ⫽ 9) and the peak height (2.39 ⫻ 10⫺4 ⫾
0.46 ⫻ 10⫺4 vs. 3.31 ⫻ 10⫺4 ⫾ 0.55 ⫻ 10⫺4, P ⫽ 0.01, paired
t-test, n ⫽ 9) of the C2-WFR increased significantly after
topical application of NGF (see Fig. 3 “augmentation” for the
depiction of this specific plasticity; see also Fig. 4).
To determine that the NGF-induced plasticity of the C2WFR in the non-depleted group was significantly different
from the lack of change in the C2-WFR induced by NGF in the
depleted group, we also compared changes in the area at
half-height and peak height between depleted and non-depleted
rats (Figs. 5 and 6). The change in the area at half height of the
C2-WFR after NGF application was significantly larger in
non-depleted versus depleted rats (36.7 ⫾ 11.2% vs. ⫺0.4 ⫾
7.5%, P ⬍ 0.05, t-test, n ⫽ 17). Likewise, the change in the
peak height of the C2-WFR after NGF application was significantly larger in non-depleted versus depleted rats (37.6 ⫾
10.6% vs. 0.5 ⫾ 8.4%, P ⬍ 0.05, t-test, n ⫽ 17). In summary,
if the cortex was depleted of ⬎35% of BFCS fibers within the
C2-WFR, then topical application of NGF to the cortex did not
induce the rapid plasticity of the C2-WFR observed in normal,
non-depleted cortex.
Last, we examined the time course of the NGF-induced
plasticity of the C2-WFR in the non-depleted rats. For the
depleted rats, topical application of NGF did not result in any
significant changes in either the area at half-height or the peak
height at any time bin measured (ⱕ100 min postapplication,
1-way repeated-measure ANOVA, n ⫽ 8; Fig. 7). For the
non-depleted rats, both the area at half-height and the peak
height of the WFR were significantly different from preappli-
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N. PRAKASH, S. COHEN-CORY, S. PENSCHUCK, AND R. D. FROSTIG
FIG. 2. Retrograde labeling studies and
cholinergic fiber depletion quantification.
Bottom: DiI injection into the supragranular
layers of the C2-whisker functional representation yields retrogradely labeled cells predominantly in the ipsilateral nBM. Coronal
section located –1.4 mm posterior from
Bregma. Right: schematized; left: stained for
acetylcholine esterase [AChE; brain atlas
picture: adapted from (Paxinos and Watson
(1986)]. The location of the DiI injection site
within the C2-whisker functional representation (WFR) supragranular layers is depicted
as a red spot under the red arrowhead (shown
in a color coronal section stained for CO
located ⫺3.0 mm posterior from Bregma).
Red dots grossly schematize that the ventral
portion of the globus pallidus (GP) had the
highest density of retrogradely labeled cells;
however, similar cells were seen just ventral
in the substantia innominata (S.I.) as well as
a few scattered cells nearby in the internal
capsule (IC). This cell cluster between GP,
IC, and S.I. is also known as the nBM. Inset:
a photomicrograph of a DiI-labeled cell body
(diameter: 20 ␮m) in the nBM after DiI
injection. Middle: composite images of coronal sections stained for AChE for control
cortex (left) and 192 IgG-saporin injected
cortex (right) from one rat at ⫻10 magnification showing the cortical layers I–VI. Note
that typically AChE staining revealed fibers
5 times as abundant as ChAT-IR fibers and
15 times as abundant as TrkA-IR fibers
(compare with Figs. 1 and 4). These slices
were adjacent slices the CO-stained slices
(see bottom), which enabled the depiction of
the location of the C2 barrel (green outline).
Top: [times}20 magnification of the same
slices from middle. Each yellow dot overlies
a fiber crossing (dots enlarged for visibility
in this figure) through 100-␮m lines drawn in
layer I and layer IV above and within the
C2-barrel. Right: the AChE-fiber-depleted
cortex after local injection of 192 IgG-saporin. In this case, the AChE-fiber depletion
was 80%.
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MECHANISMS UNDERLYING NGF CORTICAL PLASTICITY
431
FIG. 3. Depiction of potential plasticity
of a WFR. A WFR can change in area and/or
height. The central 3-dimensional-rendered
image is a typical pattern of activity for a
normal WFR with green depicting baseline
activity and the red peak depicting the maximal activity. A WFR can change after manipulations that induce cortical plasticity.
For example, the top right image depicts an
increase in height and area of a WFR, which
we term “augmentation,” such plasticity was
observed after NGF application (Prakash et
al. 1996a,b) and nicotine application (Penschuck et al. 2002). Other types of plasticity
have also been observed in WFRs, such as an
“attenuation” after BDNF application
(Prakash et al. 1996a,b) and an “expansion”
after carbachol application (Penschuck et al.
2002).
cation baseline at the time bin of 26 –50 min in the presence of
NGF (1-way repeated-measure ANOVA, post hoc Dunnett’s
test, P ⬍ 0.05, n ⫽ 9; Fig. 7). After this time bin, the values
remained elevated but were not statistically different from
baseline.
In general, the timecourse of NGF’s plasticity of a WFR
appears to be somewhat slower in rats with durotomies reported here versus rats with durectomies previously reported
(Prakash et al. 1996b). The slower time course of the NGFinduced plasticity of a WFR may be related to a reduced amount
of NGF reaching layer 1 through the durotomies compared with
durectomies and/or a longer amount of time required to attain a
sufficient concentration of NGF within layer 1.
Detection of exogenous NGF penetration to the cortical
tissue
To determine how well NGF penetrated the cortex through
the dural slits and holes (durotomies), NGF-IR was assessed in
five rats (Fig. 8). We found that NGF-IR was detected in layer
I (40 – 80 ␮m), and thus NGF-IR was detected at a depth that
was about one-third the depth of preparations previously reported with durectomies (Prakash et al. 1996b). Additionally,
NGF-IR was generally localized in the cortex to the site diJ Neurophysiol • VOL
rectly under the slits in the dura. It is likely that NGF spread
beyond the dural slits, but low-level NGF-IR throughout the
pial layer all over the brain obscured our ability to quantify
immunoreactivity at depths of less than ⬃25 ␮m below the pial
surface.
DISCUSSION
Summary of results
Several experiments were reported here that tested the hypothesis that the BFCS is involved in mediating NGF’s rapid
cortical plasticity. First, we found that the projections of the
BFCS can target a single WFR in the cortex and are mostly
located in the nBM. Second, we found that TrkA-IR fibers are
colocalized with ChAT-IR fibers in the adult rat barrel cortex
that likely arise from the BFCS and therefore that by binding
to TrkA, NGF has the potential to modulate acetylcholine
release from the BFCS. Finally, we found that depletion of
⬎35% of the AChE fibers that innervate the C2-WFR prevented the rapid NGF-induced plasticity of the C2-WFR after
topical application of NGF to the cortex. Overall, these experiments implicate the BFCS as necessary for NGF-induced
cortical plasticity.
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N. PRAKASH, S. COHEN-CORY, S. PENSCHUCK, AND R. D. FROSTIG
recent study on the role of NGF in plasticity of the developing
visual cortex led to the same conclusions (Rossi et al. 2002). In
this study, cholinergic afferents to the visual cortex were
destroyed by application of the excitotoxic drug quisqualic acid
to the BF. Afterward, using Western blot, TrkA and p75
expression was dramatically but not completely reduced in the
cortex, suggesting that in the developing rat these receptors are
located on predominantly cholinergic terminals. Taken together, these results generalize the anatomical connections
between NGF and the BFCS to both the developing and the
adult cortex.
NGF/basal forebrain cholinergic system functional
interactions
FIG. 4. Depleting the cortex of ⬎35% of AChE fibers prevents NGFinduced augmentation. Top: 3 wk after cortical 192 IgG-saporin injection and
immediately after the imaging session, AChE fiber depletion was calculated by
counting fiber crossings (see Fig. 2). This graph depicts the average number of
fiber crossings in layer 1 and 4 for the uninjected control hemisphere and the
192 IgG-saporin injected hemisphere. {, the 6 rats with ⬎35% AChE fiber
depletion; }, the 3 rats that had 0 –25% depletion. Bottom: this graph depicts
that in cortex with AChE fiber depletion of ⬍25%, NGF application augmented a WFR (}), whereas in cortex with AChE fiber depletion ⬎35%, NGF
application did not augment a WFR ({).
One way to test whether the BFCS is implicated in NGF’s
plasticity of a WFR is to directly lesion the BFCS with a
specific toxin, such as 192 IgG-saporin, to detect whether NGF
can still augment a WFR in the lesioned rat. An alternative way
is to pharmacologically block the cholinergic receptors located
on the cortical BFCS projections by topical application of a
general cholinergic antagonist and detect whether NGF can
still induce rapid plasticity of a WFR in such a pharmacologically blocked cortex. Unfortunately, there is no single general
antagonist for both muscarinic and nicotinic cholinergic receptors. Therefore to perform properly such an experiment, at least
two general antagonists are needed prior to the application of
NGF. This is a complicated experiment because the time
courses of the antagonists actions after topical application to
the cortex is different as we previously demonstrated for the
topical application of muscarinic and nicotinic receptor agonists (Penschuck et al. 2002). Thus a general cholinergic blockade of the cortex is challenging, and therefore the interpretation
of the effect of NGF application is questionable under such
conditions (Penschuck et al. 1999).
Cytochemistry of basal forebrain projection cells
and TrkA cortical fibers
The basal forebrain contains at least two major projection
neurons that have fibers in the cortex: IR (GABAergic) and
ChAT-IR (cholinergic). The cholinergic fibers are the best
candidates for also being the TrkA-IR fibers we observed in the
barrel cortex, because previous work has shown that most of
the abundant ChAT-IR cell bodies in the basal forebrain are
also p75-IR/TrkA-IR (Sobreviela et al. 1994). While there is no
question that BFCS cells also express TrkA, there is still no
direct evidence that their projection fibers in the cortex coexpress ChAT and TrkA. This lack of direct evidence may be
related to a low sensitivity of TrkA antibodies (Sobreviela et al.
1994) and even some ChAT antibodies at detecting antigen in
the relatively thin cholinergic fibers. Nevertheless as we were
able to detect TrkA fibers in the cortex, co-localization studies
with TrkA and ChAT (or VAChT) were then performed. We
found that while TrkA-IR fibers were less abundant than ChAT
fibers, they were also ChAT-IR (or VAChT; Fig. 1). Because
TrkA-IR fibers were found to be ChAT-IR (or VAChT-IR),
this provided direct anatomical evidence that NGF can act on
cholinergic fibers in the barrel cortex. It is interesting that a
J Neurophysiol • VOL
FIG. 5. NGF induces significant plasticity of a WFR in nondepleted cortex
(dark triangles) but not in AChE depleted cortex (white dots). Small symbols
represent changes from individual rats, whereas large symbols depict the
averages and SE for the 2 groups. These 2 groups were statistically different
from each other both for area at half-height and for peak height (see text for
details).
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433
FIG. 6. NGF induces significant plasticity of a WFR in
normal cortex (■, F, }, Œ, Š, ) but not in AChE depleted cortex
(䊐, E, {, ‚, ƒ). These graphs depict the actual values, not
percent normalized (as in Fig. 5), for the peak height and area
at half-height values in individual rats before and after NGF in
AChE-depleted and non-depleted cortex. Dark lines depict the
average trend for each graph.
Using the 192 IgG-saporin lesion method described in the
preceding text, we found that in rats with depletion of ⬎35%
of the AChE fibers that innervate the C2-WFR, topical NGF
application did not induce WFR plasticity, thus supporting the
FIG. 7. Time course of NGF’s plasticity in AChE-depleted vs. non-depleted cortex. 1, the average area at half-height (top) or peak height (bottom)
of a WFR before NGF application. 䊐 and ■, the averages at successive time
bins in the continual presence of NGF. In cortex with depleted AChE fibers
(left, 䊐) NGF did not increase either the area at half height or the peak height
even after 100 min of application. In contrast, in non-depleted cortex (right, ■),
NGF significantly increased the area at half-height and peak height ⬃30 min
after its topical application to the cortex. Error bars denote SE; *, P ⬍ 0.05 vs.
preapplication time bin.
J Neurophysiol • VOL
hypothesis that the BFCS is necessary for the mediation of
NGF-induced rapid plasticity of a WFR. Additional support for
functional interactions between NGF and the BFCS has been
suggested from research on the developing visual cortex of the
rat. Pesavento and colleagues have demonstrated in the developing rat visual cortex that NGF modulation of LTP is mediated by the cholinergic system (Pesavento et al. 2000). In
addition, NGF-induced increase of ACh and glutamate release
from cortical synaptosomes was strongly impaired after elimination of the cholinergic afferents to the visual cortex (Rossi
et al. 2002). Taken together, these results generalize the functional interactions between NGF and BFCS as part of an
underlying system important for cortical plasticity in both the
developing and the adult cortex. This conclusion, however,
seem at odds with a recent study that used transplants of
NGF-secreting fibroblasts to restore a reduction stimulusevoked activity in barrel cortex of the adult rat (Rahimi and
Juliano 2001). These authors demonstrate that NGF secreted
from the grafted fibroblasts could restore a reduction of
evoked-activity in a barrel cortex depleted from cholinergic
projections from the BFCS. The restoration of stimulus-evoked
activity happened in spite of the fact that the cortex was
depleted of ⬎80% of AChE. These observations led the authors to conclude that NGF can directly affect cortical activity
without the presence of the cholinergic system. The explanation for the apparent difference in conclusions may relate to
many technical differences on how the experiments were performed and analyzed. Moreover, there are major differences in
what was studied in each experiment. Rahimi and Juliano
studied the ability of chronic NGF delivery to restore normal
cortical activity in a cortex with ⬎80% AChE depletion, while
we demonstrated the lack of ability of acute NGF application
to induce transient plasticity in a cortex devoid of ⬎35% of its
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N. PRAKASH, S. COHEN-CORY, S. PENSCHUCK, AND R. D. FROSTIG
FIG. 8. NGF penetrates the cortex through holes or slits
in the dura and arachnoid mater (durotomy). Left: NGF-IR
(red arrows denote obvious penetration areas) is seen as
green in this fluorescent photomicrograph of the 1st 60-␮m
tangential section from the pial surface in the barrel cortex.
Inset: the black and white image is of the whole cortex
during the imaging session. The thin black ovals are the
sites of the 3 dural slits; a 4th small hole was also made in
this rat at the red arrow. NGF-IR through that hole is
shown here. The dark black circle approximates the area at
half-height border of the C2-whisker functional representation. The white arrowhead points to a reference surface
vein in both the inset and the photomicrograph. Right:
NGF-IR was detected under a dural slit as green in these 2
fluorescent photomicrographs of 1 40-␮m coronal section
in the barrel cortex of a different rat. The right image
shows the mediolateral extent of visible NGF-IR under a
dural slit (between red arrows). The left image is a higher
power image taken from the region indicated by the area
outlined in white and demonstrates that in this rat NGF-IR
was detectable ⬃50 ␮m below the pial surface. Control
slices not shown here had staining patterns similar to the
regions outside the red arrows.
AChE fibers. Finally, a recent study using a novel antibody to
TrkA reported the existence of TrkA-IR on ChAT-IR neurons
in layers II-III and layers V–VI in the rat visual cortex (Tropea
et al. 2002). If these finding could be replicated in the somatosensory cortex of the adult rat and the specificity of this novel
antibody further verified, it may help in clarifying the differences between ours and Rahimi and Juliano’s studies. In particular, if there are TrkA-IR and ChAT-IR neurons in somatosensory cortex in different cortical layers, there may be different layer-dependent effects. In Rahimi and Juliano’s study, the
fibroblast grafts were placed in the deep layers of the cortex
(1.1-mm depth), while our findings where occurring as NGF
was penetrating layer I (see following text).
There are at least two other potential ways to complement
our studies to obtain a better insight for the role of the BFCS
system in NGF’s ability to induce rapid plasticity of a WFR.
The first method would be to stimulate the BFCS and observe
whether WFR’s plasticity is identical to NGF’s induced plasticity. However, it is currently not possible to specifically
stimulate only the BFCS within the BF. Nevertheless, stimulation of the BF (which includes cholinergic, GABAergic, and
other components) (Dykes 1997) in the rat typically induces
enhancements of neuronal activity (Jimenez-Capdeville et al.
1997; Metherate and Ashe 1991, 1993). Although it remains to
be shown, such enhancements at the neuronal level could, in
principle, underlie the NGF-induced plasticity of a WFR. The
second way to test whether activation of the BFCS is implicated in NGF’s-induced plasticity of a WFR is to pharmacologically increase acetylcholine receptor activity. Such experiments are reported elsewhere (Penschuck et al. 2002) and used
a protocol similar to the protocol reported here, but instead of
NGF, cholinergic agonists such as carbachol or nicotine were
topically applied to the durotomized cortex. In these experiments, both nicotine and carbachol caused a transient increase
in the area of WFR similarly to NGF. However, NGF and
nicotine had identical time courses for this increase, whereas
carbachol had a slower time course. In addition, nicotine and
NGF both augmented a WFR by increasing the height of the
WFR in addition to the expansion of area, whereas carbachol
only expanded the area of a WFR without the increase in height
(compare augmentation to expansion in Fig. 3 for the depiction
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of both cases). Overall, these results are consistent with the
hypothesis that activation of acetylcholine receptors could explain WFR plasticity. They also suggest that NGF’s rapid
plasticity could be mediated predominantly through nicotinic
receptors.
Is ngf rapidly inducing plasticity by activating nicotinic
receptors in cortical layer I?
Previously, we showed that at the time point when the
NGF-induced plasticity of a WFR was maximal, NGF penetrated layer I of the somatosensory cortex (Prakash et al.
1996b). In the current study, the more limited penetration NGF
further suggests that topical application of NGF can affect
evoked cortical activity by inducing acetylcholine release from
BFCS projection axons within layer I. Assuming that the
sensitivity for the detection of the NGF using immunohistological techniques is optimal, the correlation between the penetration and plasticity could be interpreted in the following
way. NGF binding to BFCS projections within layer I causes
enhancement of ACh release. Indeed layer I contains the highest laminar densities of ACh axons and varicosities originating
from the BFCS (Mechawar et al. 2000), thus providing a rich
substrate for NGF action. The target of the cholinergic fibers in
layer I could be the apical dendrites of pyramidal and bipolar
cells from layer II to V that extend to layer I (reviewed by
Nieuwenhuys 1994) or direct activation of neurons located
within layer I.
One clue to the type of cholinergic target of NGF is its time
course. NGF’s plasticity time course and its characteristics
(augmentation) seem identical to nicotine’s (Penschuck et al.
2002). These findings suggest that NGF’s-induced rapid plasticity acts predominantly through activation of nicotinic cortical receptors within layer I. This suggestion is supported by
recent findings that demonstrate that although nicotinic receptors can be found in all layers of the adult rat somatosensory
cortex, they are heavily concentrated in layer I (Levy and Aoki
2002). Indeed, recent evidence demonstrates directly that ACh
affects neuronal activity in layer I through nicotinic receptors
by showing that almost all of layer I interneurons can be
excited by ACh nicotinic agonists. Furthermore, by activating
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MECHANISMS UNDERLYING NGF CORTICAL PLASTICITY
selectively the nicotinic receptors of layer I interneurons, it was
demonstrated that the predominant role of these interneurons is
to inhibit layer II and III nonpyramidal cells, most likely
GABAergic interneurons, and thus probably cause a disinhibition of cortical networks (Christophe et al. 2002). These authors further demonstrate that some of layer I interneurons
have indeed direct axonal projections to cortical layers II and
III. Indirect activation of layers II and III from layer I has also
been demonstrated. Stimulating layer I has been found to
induce LTP in layers II/III of the cortex indirectly via the
cholinergic processes that also reach layer I (Hess and Donoghue 1999). Taken together, cholinergic activation of layer I by
NGF can cause large-scale cortical plasticity in deeper cortical
layers through direct and indirect mechanisms that could act in
concert and seem to be the result of activation of layer I
nicotinic receptors.
Modulatory versus trophic effects of NGF
The presence of NGF in the adult cortex is thought to serve
long-term processes, such as survival and maintenance of
neurons, as an extension of its role during cortical development. These processes occur after NGF binds to TrkA and/or
p75 (reviewed in Bothwell 1995; Kaplan and Miller 1997).
NGF is rapidly internalized on binding to p75 (Kiss et al. 1993)
and/or TrkA (Grimes et al. 1997; Zapf-Colby and Olefsky
1998) into a vesicle that is transported back to the cell body
where its trophic actions occur via second-messenger cascades
within the cell body and nucleus (recently reviewed in Sofroniew et al. 2001). However, initially on binding, and
throughout the transport process, within this vesicle, the NGF/
receptor complex actively induces second-messenger pathways. Moreover, recently evidence has accumulated to suggest
that the signals induced by NGF may play other roles within
the cortex that result in more immediate effects such as increasing synaptic efficacy. For example, in slices of rat visual
cortex NGF induced a rapid increase in the height of impulseevoked excitatory postsynaptic currents through AMPA and
NMDA receptors, within 5–15 min after NGF application
linking NGF action to fast modulation via glutamate release
(Carmignoto et al. 1997). The authors speculate that the NGF
action is mediated presynaptically through severed cholinergic
nerve projections in the slice. In addition, adult cortical cell
cultures showed rapid (within 10 min) activation of calciumdependent potassium channels by NGF (Holm et al. 1997).
Taken together, these results illustrate that NGF is capable of
inducing plasticity of adult neurons on several levels. First,
initially on binding to TrkA, NGF induces acute changes in
cortical activity through changes in synaptic efficacy; such
changes may continue to be induced while the NGF/receptor
complex is transmitted back to the cell body. Once the complex
reaches the cell body, there are also induced changes in protein
expression. Hence in adult rat brain, NGF can rapidly augment
evoked sensory activity by modulating activity within the
BFCS axons, as well as maintaining the survival of the BFCS
cells.
NGF-induced plasticity: a proposed model for cortical
modulation of a diffuse modulatory projection system
The cortex is generally portrayed as a passive recipient of
modulatory systems projections including cholinergic, dopaJ Neurophysiol • VOL
435
minergic, noradrenergic, serotonergic, and histaminergic diffuse systems. Our findings implicate the cortical projections of
the BFCS in the plasticity induced by NGF and therefore show
that under certain conditions, the sensory cortex is not just a
passive recipient of the cholinergic projections but an active
participant in modulation of their activity, a modulation resulting in a large-scale plasticity of a WFR. Therefore by enhancing release of NGF, the cortex can transiently augment sensory
representations like WFRs by recruiting additional neurons to
respond to a stimulus, resulting in the expansion of the area of
a representation, and by enhancing the strength of their responses, resulting in the increased height of the representation.
It will be interesting to find out whether such cortical modulation of a diffuse modulatory system is unique to the cholinergic system or whether the cortex can also similarly modulate
other diffuse modulatory systems. An interesting prediction of
such a model is that the cortical outcome of NGF-induced
plasticity should be different from the outcome expected from
directly enhancing BF activity (i.e., a direct BF stimulation
experiment). In the case of NGF-induced plasticity, the cortex
modulates only on the BFCS projection fibers within the cortex. In contrast, directly stimulating the BF enhances unselectively the activity of all the different systems that reside within
the BF (including the BFCS) and therefore would result in a
different cortical activation pattern compared with the selective
enhancement of the cholinergic projections inside the cortex
itself.
ACKNOWLEDGMENTS
We thank J. Lee, and J. Hsu for technical assistance, Drs. R. Metherate, D.
Polley, and Cynthia Chen-Bee for helpful discussions, and Genentech, Inc., for
the generous gifts of NGF and NGF-antibodies.
Present addresses: N. Prakash, Dept. of Neurology, University of California,
Los Angeles, California 90095-6975; S. Penschuck, Neuropharmacology
Dept., H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark.
GRANTS
This work was supported by the fellowships from the University of California Irvine Medical Scientist Training Program and the American Heart
Association (N. Prakash) as well as National Institute of Neurological Disorders and Stroke Grants NS-39760 and NS-34519 and National Science Foundation Grant IBN-9507936 (R. D. Frostig).
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