1 AG Entwicklungsneurobiologie ND 6/72, Fakultät für Biologie,
Ruhr-Universität, 44780 Bochum, Germany
2 Institute for Neurophysiology, CNR, 56100 Pisa, Italy
* Author for correspondence (e-mail: wahle{at}neurobiologie.ruhr-uni-bochum.de)
Accepted 21 October 2002
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SUMMARY |
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Key words: BDNF, NT-4/5, NGF, LIF, TrkB, TrkC, Rat visual cortex, synRAS-TG mice
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INTRODUCTION |
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However, a factor that prevents LGN atrophy after monocular deprivation is
not necessarily the factor that neurons depend on during development. For
instance, a factor could act at the cortical level preventing the ocular
dominance shift as is the case for NT-4/5
(Lodovichi et al., 2000;
Gillespie et al., 2000
),
thereby allowing LGN neurons to maintain their normal connections and size
without this factor being trophic for LGN neurons. Furthermore, it is unclear
whether the effect of cortical NT-4/5 (and other neurotrophins) on thalamic
neurons is purely mediated by retrograde transport, since LGN is also target
of a corticothalamic projection.
To clarify the issue of the effects of cortical neurotrophins on thalamic
neuron development we analyzed LGN soma size development after intracortical
infusion of neurotrophins in rats with normal vision. The choice of the rat
was prompted by the wealth of molecular data present for the action of
neurotrophins in rat visual system
(McAllister et al., 1999) and
by the fact that the action of all four neurotrophins on ocular dominance
plasticity and visual cortical neuron electrical activity have been compared
in this species (Lodovichi et al.,
2000
). Cortical layer IV and VI neurons and synRAS transgenic mice
were analyzed to assess whether neurotrophin action on LGN neurons was
secondary to an action on their cortical target neurons. To test whether
anterograde transport of neurotrophins may contribute to soma growth, we
analyzed the superior colliculus (SC) taking advantage of the fact that
neurons in the ipsilateral stratum griseum superficiale (SGS) of the SC
receive only corticofugal projections. We also investigated the effects of the
cytokine leukemia inhibitory factor, LIF, because we recently found that
exogenous LIF has effects similar to NT-4/5 on neuropeptide Y mRNA expression
in the neocortex, while the endogenous LIF expression is negatively regulated
by thalamic afferents (Wirth et al.,
1998a
; Wahle et al.,
2000
).
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MATERIAL AND METHODS |
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Osmotic minipumps (pumping rate 0.5 µl/hour for about 6-7 days) were
filled with: mouse NGF1 µg/µl (2.5 S, kind gift from Dr D. Mercanti);
human recombinant NT-4/5 1 µg/µl (Regeneron); human recombinant BDNF 8.3
µg/µl (Regeneron); LIF 0.08 µg/µl (Alomone Labs, Israel); and
cytochrome C 8.3 µg/µl (Sigma) as control for nonspecific effects of the
infusion protocol. The efficacy of the LIF batches was proved in organotypic
cultures in the assay previously described
(Wirth et al., 1998a). Factors
were either infused from P12-20, which is the beginning of the critical period
in rat (Fagiolini et al.,
1994
), or from P20-28 (peak of the critical period) and animals
were either killed at the end of the infusion period or survived until P45
(end of critical period) in order to analyze long-term effects
(Table 1).
In addition, eight age-matched pairs of wild-type and synRAS-TG mice
(heterozygous line 50) were analyzed at P13 (2), P30 (1), P40 and 3 months (5)
of age. These transgenic mice have activated valin-12 mutant p21 ras under
synapsin-I promoter control and were used previously to obtain our
morphometric data of neocortical pyramidal neurons
(Heumann et al., 2000).
Histology
Animals were killed with an overdose of pentobarbital and transcardially
perfused with saline solution followed by 4% paraformaldehyde in 0.1 phosphate
buffer, pH 7.4. Brains were postfixed for 2 hours and cryoprotected in 25%
buffered sucrose overnight. The right hemispheres were marked by cuts in the
ventral cortex and brain base. Blocks containing cortex, LGN and SC were
frozen in Tissue Tek. Serial coronal sections of 30 µm thickness were cut
with a cryostat. One series of sections was mounted on gelatincoated slides
and stained with thionin. Alternating series were processed for
immunohistochemistry and in situ hybridization employing previously described
protocols (Wirth et al.,
1998a). Immunohistochemistry was performed with antibodies against
the Ca2+-binding proteins parvalbumin (PV, 1:1000, Swant) and
calbindin D28K (CB, 1:1000, Swant), and the pan-neuronal marker NeuN (1:1500;
Chemikon) then developed by the ABC-horseradish peroxidase method using
diaminobenzidine as the chromogen. For in situ hybridization, digoxigenin-UTP
or biotin-UTP (Roche) labeled antisense riboprobes were synthetized by in
vitro transcription from linearized plasmid cDNA encoding GAD67 (Gad1), GAD65
(Gad2), LIF ß-receptor, TrkB kinase domain and TrkC. Hybridization, color
development and fluorescent double-labeling was performed as described
previously (Wirth et al.,
1998b
; Gorba and Wahle,
1999
).
Analysis
In thionin-stained sections of LGN, SC and visual cortex, all neuronal
somatic outlines (nucleus present in the plane of the section) in several
randomly chosen fields of interest were reconstructed at 1000x
magnification with a camera lucida. In the LGN, neuronal somata in at least 10
such fields throughout the nucleus were reconstructed. Since we did not employ
a retrograde tracer to select for thalamocortical relay neurons, we sampled
all neurons. In the SGS we reconstructed `total' neurons in thionin-stained
sections, and CB-immunoreactive and PV-immunoreactive subsets from
ABC-horseradish peroxidase-stained sections. In the visual cortex,
thioninstained neurons were sampled in layers IV and VI of area 17 starting
about 0.5 mm lateral to the infusion site boundary. The drawings were
digitalized and soma area was determined. For every hemisphere at least 150
somata per animal were reconstructed from at least 3 non-adjacent sections in
many cases by operators familiar with soma reconstruction but blind with
respect to the hemisphere analyzed or factor infused. Ipsilateral (left
infused hemisphere) and contralateral (right untreated hemisphere) values were
compared by a non-parametric rank sum test (Mann-Witney U-test; significance
level accepted P<0.001. In addition, KS-tests and ANOVA were run.
Animals were first analyzed separately by comparing ipsilateral to
contralateral values. To construct the size frequency histograms the data rows
of ipsi- and of contralateral hemispheres, respectively, of animals of the
same age and treatment were pooled. The size shifts observed in the individual
animals were indicated as `percentage median shift' in small insets in the
figures while the median shifts for the pooled data were given in the text
together with the total cell numbers analyzed. Positive shifts indicate a
larger ipsilateral side, negative shifts indicates a smaller ipsilateral side.
The cytochrome C-infused control animals delivered the biological variability,
it was found to be no larger than ±5% median size shift.
Lack of published information on Trk receptor expression in SC cell types
required single- and double-labeling techniques. In defined regions of
interest in the SGS we counted the total number of neurons, using NeuN as a
marker, the number of GAD65 (Gad2), TrkB (Ntrk2) and TrkC (Ntrk3) mRNA
expressing neurons, the CB- and PV-immunoreactive neurons, and the proportion
of CB- and PV-immunoreactive neurons expressing GAD65, TrkB and TrkC mRNA. The
overlap of GAD65 and TrkB mRNA was analyzed with double-in situ hybridization
with a DIG-UTP-labeled GAD riboprobe and a biotin-UTP-labeled TrkB riboprobe
(Gorba and Wahle, 1999).
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RESULTS |
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BDNF infusion did not induce somatic growth in LGN
(Fig. 1B, 516 ipsi/520 contra
cells, MWU-test P>>0.05; mean size shift -0.3%, not
significantly different form cytochrome C data, one-way ANOVA Tukey post-hoc
test). Immunostaining for the infused neurotrophins has revealed the diffusion
range (Lodovichi et al., 2000)
indicating that the lack of effect of BDNF on thalamic neurons cannot be
attributed to a failure of diffusion of BDNF at cortical level. Furthermore, a
strong effect of BDNF on cortical expression of neuropeptide Y
(Engelhardt et al., 2001
) and
on the soma sizes of cortical neurons was detected in these animals throughout
the entire area 17 (see Fig.
5).
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In contrast, the infusion of NT-4/5 (Fig. 1C; 661 ipsi/638 contra cells) and NGF (Fig. 1D; 543 ipsi/581 contra cells) clearly induced somatic growth. Neurons in the LGN ipsilateral to the infused cortex displayed dramatic shifts towards larger sizes. The mean size shift with NT-4/5 was +19.2% and with NGF +14.9% (both MWU-tests P<0.0001; significantly different from that with cytochrome C, one way ANOVA P<0.0001, Tukey post hoc test). Thus, at the peak of the critical period thalamic neurons in rats with normal vision responded selectively to an increase in cortical NT-4/5 and NGF.
NT-4/5 accelerates LGN soma size development
To obtain a more complete picture of NT-4/5 action on LGN development we
compared the effects of cortical infusion at P20-28 with those of an infusion
at the beginning of the critical period, P12-20. This early NT-4/5 infusion on
LGN soma size was also effective (Fig.
2A; 502 ipsi/540 contra cells, MWU-test P<0.0001; mean
size shift +9.6%; different from that with cytochrome C and from NT-4/5 at
P28, one way ANOVA P<0.0001), although slightly less than
infusions at the peak of the critical period at P20-28
(Fig. 2B; shown again here for
direct comparison).
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It has been shown that cortical NT-4/5 can induce long lasting changes in
cortical neurochemical architecture and in cortical neurons soma size
(Wahle et al., 2000) (P. W.,
unpublished). We therefore asked whether the LGN neuron soma size increase
induced by NT-4/5 was transient or permanent. NT-4/5 was infused from P12-20
and P20-28, respectively, and animals were allowed to survive until P45
(Fig. 2C,D). In both cases,
there was no difference between ipsilateral and contralateral LGN neurons
(Fig. 2C: MWU-test
P>>0.05; mean size differences +3.2%, 343 ipsi/333 contra
cells; Fig. 2D: MWU-test
P>>0.05, mean size difference +0.6%, 535 ipsi/513 contra cells,
one way ANOVA for both data sets P>0.05). When comparing the peak
ranges of contralateral and ipsilateral LGN neurons in
Fig. 2B-D, it became evident
that the NT-4/5 infusion had precociously induced the adult size variation in
ipsilateral neurons.
Next, the medians of all ipsilateral data at P20 (NT-4/5, n=2) and P28 (NT-4/5 and NGF, combined n=7) and P45 (NT-4/5, combined n=4, untreated age-matched animals, n=2) were compiled and compared to the medians of the contralateral sides (Fig. 3). The NGF-infused animals were included because the growth effects produce by the two factors were the same. It became evident that there was a continous soma size development of contralateral LGN neurons between P20 and P45. In contrast, on the ipsilateral hemispheres NT-4/5 and NGF had accelerated this development such that LGN neurons had acquired their normal adult size already at P28 [Fig. 3; two way ANOVA side x age; age is a significant factor: size at P45 was significantly different from contra at P28 (P<0.01), whereas size at P45 was not different from ipsi at P28 (P>0.05)].
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Superior colliculus neurons grow in response to cortically infused
NT-4/5
The growth-promoting effect of NT-4/5 on LGN neurons might be mediated
solely by retrograde axonal transport
(Riddle et al., 1995).
However, neurotrophins in the visual system can be anterogradely transported,
synaptically released and transferred to postsynaptic neurons
(VonBartheld et al., 1996
;
Kohara et al., 2001
;
Spalding et al., 2002
).
Anterogradely delivered neurotrophins have functional effects because BDNF
injected into the eye affects gene expression in retinal target structures and
promotes survival of axotomized LGN neurons
(Caleo et al., 2000
). The
growth promoting effects in the LGN could thus also be influenced by
anterograde transport, since layer VI projects back to the LGN. To test
whether anterograde transport affects cell growth we analyzed neurons in an
efferent target region. The visual cortex projects to the ipsilateral SGS and
stratum opticum of the SC, anterograde label in the SC covers these upper
strata, and upper grey neurons can be synaptically activated by cortical
stimulation (Berson, 1988
;
Mize, 1996
). The corticotectal
projection becomes established at P5-7 and thus is present at the ages
investigated (Thong and Dreher,
1986
; Okoyama and Kudo,
1997
).
A direct growth response requires specific receptors. Although TrkB and
TrkC expression has been described in SC
(Allendoerfer et al., 1994;
Sobreviela et al., 1994
), the
cell type-specific expression is not known. We determined that, in the SGS,
79.3% of the NeuN-immunoreactive total neurons express TrkB mRNA, and 51.9%
express TrkC mRNA suggesting a considerable degree of coexpression of TrkB and
TrkC mRNA. CB-immunoreactive neurons represented 31% of all SGS neurons. TrkB
mRNA was detected in 87.5% and TrkC mRNA in 79% of the CB neurons suggesting
that a majority of SGS CB neurons coexpressed both Trk receptors.
PV-immunoreactive neurons represented 10% of all SGS neurons (they do not
colocalize with CB neurons) (Cork et al.,
1998
). TrkB mRNA was detected in 90.3% of the PV neurons while
TrkC mRNA was detected in only 32%. Inhibitory GAD-65 mRNA-expressing SGS
neurons represented 57% (slightly higher than previously reported)
(Mize, 1988
;
Mize, 1996
). A majority of
these small-sized somata expressed TrkB mRNA, and endogenous BDNF influences
these neurons and their inhibitory action in particular
(Henneberger et al., 2002
).
GAD-65 mRNA was detected in only 2.5% of the CB neurons, and 8% of the PV
neurons confirming the predominantly excitatory nature of these cell classes
(Mize, 1996
;
Lane et al., 1997
). TrkA mRNA
is not expressed in SC (Sobreviela et al.,
1994
) and the low-affinity neurotrophin receptor p75 is largely
confined to retinal afferents (VonBartheld
and Butowt, 2000
), although some p75 staining seems associated
with superficial tectal cells (Harvey,
1994
).
The presence of TrkB receptors in inhibitory and excitatory SGS neurons
suggested that anterogradely delivered TrkB ligands could act directly.
Therefore, total SGS neurons were sampled within the upper 200 µm in
thionin-stained material. In addition, the subset of CB neurons in the middle
SGS was sampled throughout the mediolateral extent of the SC; these neurons
give rise to the tectothalamic (including tecto-LGN) projection
(Lane et al., 1997) and do not
receive direct synaptic input from the visual cortex. The hypothesis was
clear: if cell volume regulation is mediated only by retrogradely transported
neurotrophins, SC neurons should not grow.
The two populations (total and subset) responded differentially. In control animals no significant size difference was observed between ipsi- and contralateral total SGS neurons (Fig. 4A; mean size difference +4.5%, 569 ipsi/522 contra cells). Also CB neurons were not different (Fig. 4A inset; mean size difference +1.0%, 330 ipsi/340 contra cells). Cortically infused NT-4/5 induced significant size increases of the total population indicating that anterogradely delivered neurotrophins contribute to volume regulation (Fig. 4B; mean size shift of +10.6%, MWU-test P<0.0001, KS-test P<<0.05; 883 ipsi/972 contra cells). Surprisingly, neither CB nor PV neurons were significantly different (for CB: Fig. 4B inset; mean size difference +2.4%, MWU-test P>>0.05; 873 ipsi/746 contra cells; for PV: mean size difference +1.1%, MWU-test P>>0.05, 707 ipsi/609 contra cells; not shown and only evaluated in the NT-4/5 infused material). Unexpectedly, BDNF infusion caused a highly significant shrinkage of the total SGS neurons (Fig. 4C; mean size shift -11.8%, MWU-test P<0.0001, 917 ipsi/943 contra cells). Again, the CB neurons were not significantly different (Fig. 4C inset; mean size difference -2.4%, 582 ipsi/529 contra cells). NGF had no effect (not shown).
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Hypertrophy of cortical target neurons does not elicit growth of
afferent LGN neurons
It is important to understand whether the specific neurotrophin effects on
LGN neurons are related to the action of neurotrophins on cortical neurons. We
therefore assessed, in area 17, the soma size distribution of neurons in
layers IV and VI where LGN afferents terminate. Cytochrome C control had no
effect (Fig. 5A; layer IV: 357
ipsi/364 contra cells, mean size difference -0.1%, MWU-test
P>0.05; layer VI: 499 ipsi/472 contra cells, mean size difference
+1.3%, MWU-test P>>0.05). However, a striking difference
between effects in cortical layers and action on LGN development was found for
the TrkB ligands. Although BDNF was ineffective for the LGN, it produced a
dramatic size increase of neurons in layers IV and VI
(Fig. 5B). Layer IV displayed a
mean size shift of +26.2% (747 ipsi/696 contra cells, MWU-test
P>0.0001); layer VI displayed a mean size shift of +26.7 (705
ipsi/676 contra cells, MWU-test P<0.0001; both significantly
different from cytochrome C, one way ANOVA P<0.0001).
Surprisingly, the effects of NT-4/5 were age-dependent. During P20-28 infusions, when LGN neurons responded dramatically, layer IV and VI neurons did not respond with growth (Fig. 5C; layer IV: 681 ipsi/683 contra cells, mean size difference -1.8%, MWU-test P>>0.05, not different form cytochrome C; layer VI: 661 ipsi/615 contra cells, mean size difference +4.1%, MWU-test P>0.05). However, the P12-20 NT-4/5 infusions produced significant somatic growth in both layers (Fig. 5C; layer IV: 427 ipsi/452 contra cells, mean size shift +14.7%, MWU-test P<0.0001; layer VI: 300 ipsi/312 contra cells, mean size shift +17.8%, MWU-test P<0.0001). This suggests a switch in neurotrophin dependency of the two neuron types.
NGF was not effective (Fig. 5D; layer IV: 728 ipsi/737 contra cells, mean size difference +2.9%, MWU-test P>>0.05; layer VI: 400 ipsi/400 contra cells, mean size difference +1.3%, MWU-test P>0.05). This suggested that the action of NT-4/5 on LGN development is not an indirect consequence of effects at the cortical level and that BDNF is a specific and highly potent factor for layer IV and VI neurons.
To further rule out indirect effects at the cortical level, we examined
synRAS-TG mice, which overexpress constitutively activated V12-Ha
ras. Ras is a signalling mediator of neurotrophins. The transgene expression
is driven by the synapsin-I promoter and developmentally regulated, starting
to increase from the first week onwards. synRas-TG is expressed in all
cortical layers as is synapsin I (Melloni
et al., 1993), but not in thalamic sensory nuclei
(Heumann et al., 2000
). We
previously reported that synRAS-TG mice displayed hypertrophic cortical
pyramidal neurons and increased neuropeptide expression indicating that
activated p21ras acts as an `intracellular neurotrophin'
(Heumann et al., 2000
). The
question was clear: if the target neurons in the cortex grow larger, does the
afferent population react with a size increase?
Five sibling pairs of adult wild-type and transgenic animals (heterozygous line 50) aged P40 and 3 months were analyzed. Layer IV neurons displayed a dramatic hypertrophy with highly significant larger sizes in synRAS-TG mice (Fig. 6A; 750 TG/729 WT neurons, mean size shift WT versus TG +22.4%, MWU-test P<0.0001; similar size shifts occurred in layer VI, not shown). However, we found no systematic size shifts in the LGN (Fig. 6B; 749 TG/779 WT neurons, mean size difference +0.2%, MWU-test P>>0.05). This indicated that LGN neuron size was neither affected by the constitutively active p21ras in cortical neurons in general nor by the larger size of their cortical target neurons. We further analyzed 3 pairs of mice aged P13 and P30, ages comparable to neurotrophin-infused animals, and that overlapping the critical period of plasticity. However, no size shifts in the LGN were detected (Fig. 6B; 373 TG/374 WT neurons, mean size difference -1.9%, MWU-test P>>0.05) suggesting that no developmental acceleration occurs in LGN neurons at times when V12-ras expression causes the hypertrophy in cortical neuron classes. Together, these results suggested that somatic growth of the cortical target cell population (produced by BDNF or in the transgenic model) does not elicit compensatory growth of the afferent LGN neurons.
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Increase in cortical availability of LIF inhibits growth of LGN and
SGS neurons
In organotypic cortex cultures, the cytokine LIF promotes NPY mRNA
expression like NT-4/5, and the endogenous LIF expression is downregulated by
thalamic fiber ingrowth (Wirth et al.,
1998a,b
;
Wahle et al., 2000
). While
cortical neurons express LIF ß-receptor mRNA, we failed to detect it in
LGN and SC neurons (not shown). We therefore compared the effects of
intracortical NT-4/5 and LIF infusion. Unexpectedly, LIF inhibited growth
(Fig. 7). After the P12-20
infusion period the LGN neurons in the infused hemisphere were very
significantly smaller than the contralateral hemisphere
(Fig. 7A; 841 ipsi/893 contra
cells, mean size shift -9.6%, MWU-test P<0.0001; significantly
different from cytochrome C, one way ANOVA P<0.05), but without
symptoms of degeneration. Two animals were allowed to survive the P12-20
infusion period until P45. At this stage, LGN neurons were of equal size
variation (Fig. 7B; 422
ipsi/396 contra cells, mean size difference +1.4%, MWU-test
P>>0.05). This indicated that the LIF-induced inhibition of LGN
somatic growth is transient and that the neurons manage to grow to adult
control size variation after the end of the LIF infusion.
|
In addition, P12-20 LIF infusions affected cortical layer IV and VI neurons, which underwent dramatic shifts to smaller sizes (Fig. 7C; layer IV: 867 ipsi/784 contra cells, mean size shift of -19.1%, MWU-test P<0.0001; layer VI: 791 ipsi/691 contra cells, mean size shift -11.0%, MWU-test P<0.0001).
Furthermore, the P12-20 LIF infusions caused a dramatic growth delay of the total SGS neurons (Fig. 7D; 667 ipsi/649 contra cells, mean size shift -11.6%, MWU-test P<0.0001), and surprisingly now also of the tectothalamic CB neurons in middle SGS (Fig. 7D; 795 ipsi/852 contra cells, mean size shift -11.8%, MWU-test P<0.0001). However, both neuronal populations no longer showed size differences at P45 (Fig. 7D; total SGS neurons: 305 ipsi/315 contra cells, mean size difference -4.9%, MWU-test P>0.05; CB neurons: 253 ipsi/270 contra cells, mean size difference -1.4%, MWU-test P>>0.05). This indicated that the LIF-induced inhibition of SGS neuron growth is transient and recovers after the end of the infusion.
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DISCUSSION |
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Retrograde and anterograde actions of NT-4/5
The NT-4/5 effect could be due to a direct action on LGN terminals because
full length TrkB receptors are expressed by LGN neurons and their cortical
axon terminals (Silver and Stryker,
2001). Riddle et al. (Riddle
et al., 1995
) showed a retrograde action, because in their study
neurotrophins were coupled to latex beads allowing only retrograde transport.
The failure of P20-28 NT-4/5 infusion to evoke a size increase in layer IV and
VI neurons indicates that a specific trophic response of LGN neurons can be
produced in the absense of a similar effect on their target neurons. This
suggests that at the peak of the critical period of visual cortical
plasticity, layer IV/VI neurons release NT-4/5 as a trophic factor for their
afferent neurons without responding themselves to NT-4/5. However, since these
neurons responded during the P12-20 infusion period, they must have
selectively downregulated the responsiveness to NT-4/5 (but conserved the
responsiveness to BDNF) towards the peak of the critical period.
Our results on SGS neurons now change the interpretation of a solely
retrograde action. Selectively those neurons contacted by cortical fibers
(Mize, 1996;
Mize, 1988
) respond with
somatic growth, and likely the GAD-65/TrkB mRNA expressing inhibitory neurons
receiving somatic synapses are the responsive neurons. Indeed, the
corticotectal pathway exerts inhibitory actions on the activity produced by
the retinotectal pathway (McIllwain and
Fields, 1971
), and it activates SC interneurons and inhibits LTP
through GABAergic mechanisms (Hirai and
Okada, 1993
). In contrast, the tectothalamic CB neurons were not
responsive, despite the fact that these neurons also express TrkB in addition
to TrkC mRNA. These neurons receive retinal, but not cortical input, respond
to enucleation with a decline in CB immunoreactivity, and most are excitatory
(Mize, 1996
;
Dreher et al., 1996
) (present
study). The excitatory PV neurons are also targeted predominantly by retinal
input (Mize, 1996
).
Since SGS neurons do not project to cortex, this finding reveals a clear
anterograde trophic effect of NT-4/5 during postnatal development. Spalding et
al. (Spalding et al., 2002)
recently showed that ocular injections of NT-4/5 in newborn rats was
anterogradely transported and reduced cell death in the SC. Moreover, our
NT-4/5 effect appears extraordinarily cell type-specific and input-specific,
as if NT-4/5 is delivered by cortical afferents exclusively to SGS inhibitory
neurons without affecting nearby excitatory neurons. We must therefore
conclude that the strong effects on LGN development in animals with normal
vision could stem both from anterograde and retrograde actions of cortical
NT-4/5.
BDNF fails to accelerate LGN development
BDNF had no effects on LGN soma size development and cannot prevent the
structural defects caused by monocular deprivation
(Riddle et al., 1995).
However, LGN projection neurons are able to respond to BDNF derived from
another source, since BDNF injected into the eye rescues LGN neurons from
death after visual cortex ablation (Caleo
et al., 2000
). Thus, for LGN survival the loss of target-derived
cortical neurotrophins can be compensated for by an excess of retinal BDNF,
whereas cortex-derived NT-4/5 appears to play a dominant role in controling
LGN soma size development. Thus, BDNF and NT-4/5 clearly have a different
profile of action in the visual system despite the fact that both converge on
the TrkB receptor.
The failure of BDNF was not due to a lack of diffusion or biological
activity, because on cortical neuron populations analyzed concurrently in
these animals BDNF altered gene expression
(Engelhardt et al., 2001) and
induced a hypertrophy of layer IV and VI neurons throughout area 17. This is
in line with the data from the synRAS-TG mice. The lack of LGN soma growth in
these mice confirmed that boosting the size of layer IV and VI target neurons
during the critical period and in adulthood does not trigger a growth of
afferent neurons. It further suggests that the specific neurotrophin effects
on LGN development do not depend on activation of the ras pathway in cortical
neurons.
The BDNF-induced shrinkage of SGS neurons was an unexpected result which is
difficult to explain. Having identified anterograde NT-4/5 as a major mediator
for volume regulation, we suggest two scenarios. BDNF infusion might reduce
cortical NT-4/5 expression followed by a reduced delivery to SGS inhibitory
neurons contacted by the fibers, followed by shrinkage. Alternatively, BDNF is
transported anterogradely (Caleo et al.,
2000; Kohara et al.,
2001
), and the excess of cortically infused BDNF might compete
with endogeneous NT-4/5 for anterograde transport, thus reducing the amount of
NT-4/5 delivered to SGS neurons. Exogenous BDNF can apparently not compensate
for the loss of NT-4/5 in the afferent axons. However, BDNF did not shrink LGN
neurons, either because these neurons manage to aquire sufficient quantities
of NT-4/5 by retrograde mechanisms or the suggested BDNF-induced
downregulation of NT-4/5 is confined to corticotectal pyramidal neurons.
NGF accelerates LGN development
It has been demonstrated that antagonizing endogenous NGF action during
development causes shrinkage of LGN neurons
(Berardi et al., 1994). We now
show that in the rat cortical NGF, like NT-4/5, accelerates LGN cell growth.
This effect is likely mediated by activation of cortical TrkA, since the
specific TrkA-activating antibody RTA also rescues LGN neurons from monocular
deprivation-induced shrinkage (T. Pizzorusso, N. B. and L. M., unpublished)
and prevents monocular deprivation effects in visual cortex
(Pizzorusso et al., 1999
).
TrkA is not expressed by LGN neurons
(Merlio et al., 1992
;
Holtzman et al., 1995
), and
LGN neurons do not retrogradely transport cortically infused NGF or RTA
(Domenici et al., 1994
;
Pizzorusso et al., 1999
).
Rather, TrkA is expressed by cholinergic afferents
(Merlio et al., 1992
;
Sobreviela et al., 1994
;
Holtzman et al., 1995
;
Pizzorusso et al., 1999
;
Rossi et al., 2002
) and some
cortical glutamatergic terminals (Sala et
al., 1998
). NGF could act on cholinergic and glutamatergic
transmission in the visual cortex, causing the release of Trkb ligands, which
then act directly on LGN afferents. LGN fibers in cat and rat have nicotinic
receptors (Prusky et al.,
1988a
; Prusky et al.,
1988b
). An attractive hypothesis is that NGF, by increasing
acetylcholine release (Rylett and
Williams, 1994
; Sala et al.,
1998
) facilitates transmission of LGN fibers, thus stimulating
release of NT-4/5 in layers IV and VI, or alternatively increase the amount of
NT-4/5 delivered to the LGN by anterograde transport. Muscarinic m1 receptors
in turn are in layer IV neurons during the critical period
(Prusky and Cynader, 1990
);
they can stimulate the MAP kinase pathway
(Rosenblum et al., 2000
)
involved in the control of gene expression, growth and plasticity. This
scenario makes sense when considering the trophin dependency of the
thalamocortical target neurons. At the peak of the critical period layer IV
and VI neurons respond selectively to BDNF, but no longer to NT-4/5 which they
probably supply to their LGN afferents. The absence of NGF effects on SGS
neurons (at best, positive trends were observed) as compared to its
unequivocal effect for LGN neurons could suggest that the NGF-induced
mobilization of NT-4/5 is confined to the thalamocortical target layers and/or
the backprojecting layer VI pyramidal neurons.
LIF delays maturation
Specific LIF ß-receptors are expressed by cortical excitatory neurons,
and to variable degrees by interneuronal populations
(Wirth et al., 1998b), but not
by LGN or SGS neurons. LIF infusions cause cortical neuropeptide expression
(similar to NT-4/5 and BDNF) (Wahle et
al., 2000
) (P. W., unpublished), but a transient delay in soma
size development of cortical neuron populations (P. W., unpublished). LIF has
the same growth-delaying action on SGS and LGN, although both populations
recover quickly after the end of the LIF infusion. An important result is that
ingrowth of thalamic fibers in the cortex in vitro downregulates cortical LIF
mRNA (Wahle et al., 2000
):
possibly, low LIF levels might be necessary for the thalamocortical fibers to
form functional connections able to sustain their growth while increasing
cortical LIF levels could be detrimental for LGN development. One explanation
compatible with the interpretation of the cortical and subcortical effects is
that an excess of exogeneous LIF transiently reduces production, release or
action of cortical TrkB ligands, this way reducing the anterograde or
retrograde supply of the growth-promoting NT-4/5 to LGN and SGS neurons. For
instance, NGF and LIF have antagonizing roles in the maturation of olfactory
receptor neurons with LIF inhibiting differentiation
(Moon et al., 2002
). The
explanation requires LIF receptors on cortical neurons, but not on the
subcortical populations, and no axonal transport of LIF. Alternatively, the
observation that LIF induces a severe shrinkage of layer IV and VI neurons
suggests that a smaller cortical target cell population may cause a delay in
maturation of afferent neurons. Support comes from the observation that LIF
was the only factor affecting also the tectothalamic CB neurons in the SGS.
The growth delay of LGN neurons has apparently caused a growth delay in their
tectothalamic afferent population in a transneuronal way.
Conclusions
The study presents three important results. First, central neurons regulate
soma size in an age- and ligand-specific fashion. Second, NT-4/5 and NGF
accelerate LGN development in rats with normal vision while LIF delays growth.
Third, NT-4/5 mediates growth of inhibitory SGS neurons via anterograde axonal
transport. It is interesting that NGF and NT-4/5, which in rat most
effectively prevent ocular dominance shifts after monocular deprivation
(Lodovichi et al., 2000), are
also the only factors able to promote LGN development. This would suggest that
in addition to their effects on cortical targets, a direct or indirect action
on subcortical neurons is an important component of the role these factors
play in cortical plasticity. In contrast, BDNF-responsive targets clearly
differ from those of NT-4/5, and are primarily located at the cortical
level.
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ACKNOWLEDGMENTS |
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