Title: Effects of Whisker-trimming on GABAA receptors in SI Cortex
Or in adult barrel cortex
CHAPTER 1
INTRODUCTION
Sensory deprivation has been a productive approach to investigate the effects of
environmental stimuli on adult and developing brain. Whereas lack of normal excitatory inputs
leaves some cortical neurotransmitter systems unaffected (Goodman et al., 1993; Schlaggar et al,
1993), it can lead to down regulation of the gamma-aminobutyric acid (GABA)-ergic system. For
instance, the cortex of monkeys, cats, and rats shows particular changes in GABAergic
components after different types of sensory or visual deprivation. In the rat somatosensory cortex,
deprivation of whisker input results in decreases in GABAergic circuitry elements, such as the
number and proportion of GABA-immunoreactive synaptic contacts in layer IV (Micheva and
Beaulieu, 1995), glutamic acid decarboxylase (GAD) levels (Akhtar and Land, 1991), and
muscimol binding to GABAA receptors (Fuchs and Salazar, 1998).
Alterations in specific GABA receptors subunits might help understand GABAergic
function. This is the first investigation where whisker deprivation regulates specific GABAA
receptor subunits.
The Whisker to Barrel System of the Rat
The rodent somatosensory whisker to barrel pathway has several features that make it a
desirable system to study sensory deprivation. For instance, there is a 1:1 topographic relationship
1
between each of the whiskers in the rat’s face and a group of neurons that constitute a ‘barrel’ in
layer VI of Somatosensory Cortex (SI) (Woolsey and Van der Loos, 1970): each barrel responds
primarily to one principal whisker. This feature enabled the discovery of cytoarchitectonic
(Woolsey and Van der Loos, 1970; Welker and Woolsey, 1974; Van der Loos and Woolsey, 1973)
and physiological (Welker, 1971, 1976; Simons, 1978; Simons and Woolsey, 1979) effects of
whisker stimulation and/or deprivation, and to investigate the experience-dependent maintenance
of synapses (Micheva and Beaulieu, 1996), neurotransmitters (Micheva and Beaulieu, 1995), and
neurotransmitter receptors (Fuchs, 1995). Moreover, the SI cortex of rats is accessible to surgical
procedures, allowing for a variety of chemical, physiological and mechanical preparations and
manipulations, such as measurements of changes of GABA receptor subunits after topical cortical
blockade of NMDA receptors (Penschuck et al., 1999).
Sensory Deprivation and GABA A Receptors
Effects of sensory deprivation on GABAergic cortical circuitry have been widely studied.
Pioneer studies on the adult monkey’s visual system showed that depriving visual input from one
eye results in decreases of both GABA and its synthesizing enzyme GAD in the deprived cortical
neurons (Hendry and Jones, 1986). In the SI cortex of adult rodents, similar effects of deprivation
have been observed. First of all, GAD is reduced in deprived barrels after trimming whiskers for 6
weeks beginning in the adult, but not beginning in the neonate (Akhtar and Land, 1991).
Physiological studies showed that simply trimming rat whiskers leads to signs of disinhibition in
the deprived barrel neurons, such as higher spontaneous activity, and a decreased selectivity to
respond to specific angles of whisker deflection (Simons and Land, 1987). These physiological
changes remain even after allowing neonatally deprived rats to regrow their whiskers for several
2
weeks, indicating the dramatic, long-lasting effect of neonatal deprivation. What is the chemical
basis of these physiological changes? Since GABA is the main inhibitory neurotransmitter in
cortex, it was important to consider GABA and its receptors as suitable candidates responsible for
these physiological changes. Blocking GABAA receptors with the antagonist bicuculline results in
signs of cortical disinhibition (Kyriazi et al, 1996) that are similar to those from deprived barrel
cortex. Furthermore, binding of the GABA agonist [3H]muscimol, which selectively binds to
GABAA receptors, is reduced in the deprived barrels (Fuchs and Salazar, 1998). This effect was
observed in both neonatally and adult deprived rats, and was still present even after allowing the
rats to grow their whiskers for ten additional weeks after the trimming period. These overall
decreases after deprivation were suggested as a down-regulating mechanism that compensates for
the reduced sensory input (Fuchs and Salazar, 1998). Recent studies showed that whisker trimming
for 2 months, starting at birth, reduced the numerical density of both intracortical and
thalamocortical symmetrical synapses, upon inhibitory neurons in barrel neuropil (Sadaka et al.,
2003).
GABA inhibitory effects in cortex depend on the types of GABA receptors involved.
Inhibition through the GABAA receptor, a chloride channel itself, is relatively fast. These receptors,
when activated, increase the chloride conductance of the membrane, affecting transmitter release in
the presynaptic neuron, and producing hyperpolarization or shunting inhibition of the postsynaptic
neuron (MacDonald and Olsen, 1994; Xi and Akasu, 1996). Through the metabotropic GABAB
receptor inhibition is slower and involves changes in potassium and calcium permeability at
presynaptic and postsynaptic synapses (Misgeld et al., 1995; Howe et al., 1987; Deisz and Prince,
1989; Deisz et al., 1997).
3
GABA Receptor Subunits and Sensory Deprivation
GABAA receptors subunits comprise a family of at least 17 subunits (Davies et al., 1997).
Each subunit is expressed in a particular laminar pattern in SI and visual cortex (V1). For instance,
in SI and V1, the α1 subunit, which is present in the majority of the GABAA receptors, is densest in
layers III-IV (Fritschy et al., 1994). This laminar-specific expression might represent distinct
functional areas which can be differentially affected by sensory deprivation paradigms. For
instance, while monocular intravitreal injection of TTX in monkey induces a reduction in α1, β2,
and γ2 subunit mRNAs subunits levels in deprived visual cortex, it leaves levels of α2, α4, and β1
unchanged (Huntsman et al., 1994; Jones, 1997). In focal cortical malformations induced by
neonatal freeze lesions of SI, α1 and α5-GABAA subunits decrease in rat SI (Redecker, 2000).
Furthermore, electrolytic lesion of thalamus in the newborn decreases α1 in layers III-IV, but
increases α2, α3, and α5 in the same SI layers (Paysan, 1997).
When whiskers are trimmed during a critical period of early postnatal development,
stimulation of the regrown whiskers causes a degraded tuning of layer II/III receptive fields in the
corresponding deprived column (Lendvai et al., 2000; Stern et al., 2001). Similarly, plucking
whiskers from birth results in weaker responses of neurons in layers II/III and IV of the related
barrel column (Fox, 1992, 1994), but causes stronger responses from neighboring barrel columns
(Simons and Land, 1987; Fox, 1992, 1994). Similar deprivation effects are recorded in layer II/III
and IV barrel neurons after adult deprivation in rats (Glazewski and Fox, 1996; Wallace and Fox,
1999). However, it has not been determined whether there are decreases in GABAergic
components of layers II/IIII that could account for the apparent disinhibition in these layers.
GABAA receptors decrease in layer IV of deprived barrel neurons (Fuchs and Salazar,
4
1998). These effects might not only be restricted to this layer, but might be present in layers II and
III of the same deprived whisker barrel columns. Moreover, changes in GABAA receptor binding,
as assessed by autoradiography, might be paralleled by changes in GABAA receptor subunits. A
favorable candidate α1-GABAA subunit, since it is the one that predominates in layers II/III of
barrel columns (
), and is constituent of most GABAA receptors (
). In the
present study, to determine whether sensory deprivation affects GABAA receptors in layers II/III
and IV, quantitative autoradiographic tests were performed on adult whisker trimmed rats. To
determine whether the same deprivation paradigm affects the α1-GABAA receptor subunits,
immunocytochemical methods were used.
5
CHAPTER II
MATERIALS AND METHODS
Subjects
The subjects were Long-Evans hooded rats (Simonsen, Gilroy, CA), maintained on a
12:12-hour light:dark cycle, with food and water available ad libitum. Whiskers from either the
middle row C or the other rows ABDE were trimmed for six weeks.
Whisker Deprivation
Six-week-old rats had whiskers trimmed every other day for 6 weeks, ensuring that their
vibrissae were kept shorter than 1 mm. All experimental rats had the mystacial vibrissae in either
row C, rows ABDE, or all rows clipped on one or both sides of the face. During the procedure,
no anesthetic was used, and rats were hand-held with gentle restraint. These protocols were
approved by the Institutional Animal Care and Use Committee at the University of North Texas.
Histology
Unperfused rats were killed at the end of their deprivation schedules by decapitation. For
tangential sections the deprived barrel region was dissected out from the brain, and was flattened
and frozen at -44˚C with the heat dissipater of a cryostat (2800 Frigocut N, Reichert-Jung,
6
Cambridge Instruments, Deerfield, IL). Cryostat sections 16 µm thick were cut at -20˚ C
tangentially to the pial surface, and were thaw-mounted onto gelatin subbed microscope slides.
For coronal sections the whole brain was frozen in -40˚C isopentane for 5 min, transferred to 80˚C isopentane, and cut and mounted as above. Sections were air dried for 0.5 to 3 h and then
stored desiccated at -80˚C until their use for either immunochemistry or receptor
autoradiography. After receptor autoradiography, some sections were Nissl stained, to examine
whether cellular and immunological observations can be paralleled. Other sections were stained
for cytochrome oxidase (CYO) activity (Wong-Riley, 1979) to localize barrels and to analyze
any similarities between CYO activity and receptor binding.
Immunocytochemistry
Subunit-specific antisera were used to visualize GABA subunits. The α1-GABAA receptor
subunit antisera were raised in rabbit against synthetic peptides derived from mouse subunit cDNA
(generously provided by J.M. Fritschy).
Prior to the immunological procedure, slides with rat sections were transferred to a 10˚C
refrigerator for 5-10 min. Then they were dried in a slide warmer for 45 sec, immersed horizontally
for 20 min in 0.5% paraformaldehyde in 0.15 M phosphate buffer (pH 7.4) (Fristchy and Mohler,
1995) for 20 min, and rinsed for 10 min in ice-cold 0.5 M Tris-saline buffer (TBS), pH 7.6. This
and all of the following steps were performed with gentle agitation using a rocker table. Slides
were then washed in preincubation solution consisting of TBS containing 10% normal goat serum
and 0.05% Triton X-100.
Sections were and incubated for two days and two nights at 4˚C in primary antibody
solution (1:100,000) diluted in TBS buffer containing 10% normal goat serum (NGS). Sections
7
were then washed three times for 1.5 hr in TBS and incubated for 30 min at room temperature in
TBS containing 2% NGS and goat-raised anti-rabbit biotinylated secondary antibodies (Jackson
Immunoresearch Laboratories Inc., West Grove, PA) diluted 1:300 in TBS pH 7.4. After 1 hr
additional washing in TBS, sections were transferred to the avidin-peroxidase solution (Vectastatin
Elite kit; Vector Laboratories, Burlingame, CA) for 30 min, and then washed 30 min in TBS.
Sections were then stained using two equal volumes of 0.1% diaminobenzidine hydrochloride
(DAB) in 50 mM TBS. To one of the volumes of substrate solution 0.02% hydrogen peroxide was
added to the sections to start the reaction, and sections were vigorously agitated for 1-2.5 min until
the desired color was obtained. The reaction was stopped by transfer into ice-cold TBS. After three
more 10 min washes in TBS, sections were dried in a slide-warmer, dehydrated with ascending
series of ethanol, cleared in xylenes and coverslipped using DPX.
Ligand binding
GABAA receptors were assessed using [3H]muscimol (Mower et al., 1986; Schwark et al.,
1994). These same sections were previously used to examine changes in layer IV after deprivation
(Fuchs and Salazar, 1998). Sections were preincubated 20 minutes at 4oC in 0.31 M Tris-citrate
buffer, pH 7.1, and then were incubated for 40 minutes at 4oC in the buffer containing 10 nM
[3H]muscimol (12-20 Ci/mmol; DuPont NEN, Boston, MA). Sections then were rinsed twice for
30 seconds in cold buffer, dipped briefly in dH20, and dried in a stream of air. Nonspecific binding
was assessed in the presence of 1 mM GABA.
Autoradiography
8
The brain sections and tritium standards (Microscale, Amersham) were exposed
simultaneously in the same cassette to tritium-sensitive Hyperfilm (Amersham). Following 2-4
month exposure periods, the film was developed with Kodak D-19 and processed according to the
manufacturer’s instructions.
Data analysis: Immunostained and Nissl stained sections
For semiquantitative image analysis, sections were digitized using a video-based
computerized image analysis system (MCID, Imaging Research, St. Catherine, Ont., Canada).
Measures of relative optical density were obtained from all the tangential α1-GABAAimmunostained sections, from cortical layer I (whenever possible) to layer IV. Samples were taken
within a computer-generated circle over each barrel column, allowing for comparisons between
deprived and intact rows.
Measures from the readily visible barrels in layer IV were taken first. Readings from upper
layers were then obtained. Patterns in the positions of radially oriented blood vessels were used to
confirm the exact location of each barrel column. Determination of boundaries between layers
II/III and IV was aid with a light microscope, taking into consideration cellular differences
between granular layer IV and supragranular layer III, and that septa are more conspicuous in layer
IV than in layer III.
Within each section, the mean ratio of densities in deprived/nondeprived row was
determined, using rows B, C, and D only. The average ratio for each subject was then calculated
from the section means. Then the average ratio for all subjects was obtained. To test the null
hypothesis that this ratio was not different from 1, Student’s t-test (two-tailed) was used.
Experimental groups were compared with one another by using analysis of variance with post hoc
9
t-tests. The significance level was 0.05. Percent decrease within each section was calculated as the
mean for deprived barrels minus the mean for nondeprived barrels, divided by the mean for
nondeprived barrels. Standard error measurements (S.E.M.) were calculated first for each subject.
For display, images were contrast-enhanced in pseudocolor.
Data analysis: Autoradiographs
[3H]Muscimol was quantitatively analyzed using a video-based computerized image
analysis system (MCID, Imaging Research, St. Catharines, Ont., Canada). Tritium standards were
used to calibrate autoradiographic densities. Ligand binding densities were automatically
calibrated by using a best-fit equation based on the plastic tritium standards, which had been
calibrated in nCi/mg tissue wet weight based on standards made from rat brain tissue (Fuchs and
Schwark, 1993). The analysis of data was done by the same procedures as the immunostained and
Nissl stained sections.
10
CHAPTER III
RESULTS
Effects of Whisker Deprivation on α1-GABAA receptor subunit
Coronal sections from control subjects depict the normal laminar distribution for α1GABAA receptor subunit immunostaining, in which layer IV appears the darkest (Fig. 1). Adult
rats with whiskers trimmed for 6 weeks showed in tangential sections a clear decrease in optical
density immunopositive staining for α1-GABAA receptor subunit in the deprived barrel columns.
Deprived columns showed less staining than the adjacent non-deprived ones. This decrease was
larger and readily detected in layers II/III of the deprived barrel columns 6% ± 0.6 (P<0.005). The
effect in layer IV was smaller, but still significant (-3.3% ± 0.9, P<0.005, Fig. 3, Table 1). Control
subjects showed no difference between C and adjacent rows (
11
).
Nissl Staining
Identification of barrel columns boundaries was aided by the differential staining and cell
density between barrel septa and hollow. Figure 4( with coronal and tangential sections). Trimming
whiskers for 6 weeks in adult rats resulted in an overall decrease in layers II, III, and IV of -8.7%
± 0.9 (mean ± S.E.M., P<0.005) as compared to the adjacent non deprived columns. This
decrease was largest in supragranular layers II and III (-11.1% ± 1.5, P<0.005), and smaller in
layer IV (-5.6% ± 0.7, P<0.005), where the difference was less obvious. (fig 4, Table 1). In
control subjects visual inspection rendered no difference between C and adjacent rows.
GABAA receptor autoradiography
[3H]Muscimol binding showed an overall reduction of -11.0% ± 0.9 in the deprived
columns as compared to the adjacent intact ones (mean ± S.E.M., P<0.001). The decrease was
similar for layers II/III (-11.4% ± 0.9, P<0.001), and IV (-10.2% ± 0.9, P<0.005). Comparison
between these two percentages from supragranular and granular layers was non significant.
Control subjects showed no difference between C and adjacent rows.
12
Cortical Layers
[3H] Muscimol
Binding
II, III, and IV
II/III
IV
Difference (II/III) - IV
-11.0 ± 0.9***
-11.4 ± 0.9***
-10.2 ± 0.9***
n.s.
α1-GABAA receptor
subunit
Immunoreactivity
-4.9 ± 0.6***
-6.0 ± 0.6***
-3.3 ± 0.9***
*
Nissl
-8.7 ± 0.9**
-11.1 ± 1.5***
-5.6 ± 0.7***
**
Table 1. Percentage decrease in the deprived barrel rows relative to intact rows (mean ± S.E.M.)
*P<0.05;**P<0.005; ***P<0.001; n.s., not significant.
13
Effects of Whisker Trimming on Layers II,III, and IV of SI
14
% decrease on deprived vs intact columns
12
10
8
6
4
2
0
α1-GABAA receptor subunit
[3H] Muscimol Binding
Nissl
Fig 1. Effects of whisker trimming on α1-GABAA receptor subunit immunoreactivity,
[3H]Muscimol binding, and Nissl staining on deprived barrel columns. After 6 weeks of whisker
trimming in adult rats, deprived rows containing layers II, III, and IIV showed significant
decreases in all three markers (**p<0.005) compared to adjacent undeprived rows.
Each value represents the mean ± S.E.M.
14
14
Percent decrease in deprived barrel layers
12
10
8
6
4
2
0
II/III**
IV*
α1-GABAA receptor subunit
II/III**
IV*
[3H] Muscimol Binding
II/III*
IV*
Nissl
Fig X. Effects of whisker trimming on α1-GABAA receptor subunit immunoreactivity,
[3H]muscimol binding, and Nissl staining on deprived barrel columns of layers II/III, and IV of
SI of rats. The effects were larger in supragranular deprived layers II/III than in deprived layer
IV for all paradigms. For α1-GABAA receptor subunit immunoreactivity the decrease in layers
II/III was 6% ± 0.6, P<0.001, and decrease in layer IV was 3.3% ± 0.9, P<0.001. For
[3H]muscimol binding the decrease in layers II/III was 11.4% ± 0.9, P<0.001, whereas in layer
IV it was 10.2% ± 0.9, P<0.001. For Nissl staining the decrease in layers II/III was 11.1% ± 1.5,
P<0.001, and in layer IV it was 5.6% ± 0.7, P<0.001. The difference in percent deprivation effect
in supragranular versus granular layer was significant, both for α1-GABAA receptor subunit
immunoreactivity (P<0.005) and for Nissl staining (p<0.05). Each value represents the mean ±
S.E.M.
15
CHAPTER IV
DISCUSSION
Effects of whisker trimming on GABAA binding
Decreases in GABAA receptors, as found in this study, may participate in the long
lasting disinhibition in neurons of deprived barrel columns, such as the increase in spontaneous
activity, and the decrease in preference for angle of whisker deflection (Simons and Land, 1987).
This respecification of receptive fields can also be mapped using markers for neuronal activity
such as 2-deoxyglucose (Kossut et al., 1988; review in Kossut, 1992). It seems that this increased
disinhibition arises within the cortex (Simons and Land, 1994), since deprivation effects can be
avoided by cortical infusion of muscimol (Wallace et al., 2001). These cortical map modifications
seem to be compensatory, brought about by the reduction in sensory input to the brain (Siucinska
and Kossut, 1996).
Our results are in agreement with previous reports in which sensory deprivation in the adult
can modify rat somatosensory cortex (Glazewski and Fox, 1996; Fuchs and Salazar, 1998). Adult
plasticity differs from developmental plasticity in fundamental ways. In the adult, experienceregulated plasticity may be a temporal, reversible event, that involves specific changes in synaptic
strength, and that may result in synapse formation and elimination (Trachtenberg et al., 2000;
Rausell and Jones, 1995; Huntley, 1996). These changes could be expected primarily in the
16
cortico-cortical circuitry (Armstrong-James et al., 1994). It is known that after 21 days of age rats
achieve the adult number of synapses per neuron (Micheva and Beaulieu, 1996). During
development, however, these changes might be irreversible (Lendvai et al., 2000), and linked to
changes in collateralization and arborization of thalamocortical fibers (Antonini and Stryker,
1993).
Whisker deprivation changes the excitability of neurons in cortex in a layer-specific
manner. Layer IV becomes more sensitive to whisker deprivation around postnatal day 7 (Fox,
1992), and remains plastic in the adult (Huang et al., 1998; Goldreich et al., 1999). Layers II/III
reach mature receptive field organization around postnatal day 14 (Stern et al., 2001) and also
remain plastic in adulthood (Armstrong-James et al., 1994; Glazewski and Fox, 1996; Lendvai et
al., 2000, Sheperd et al., 2003). For instance, [14C] 2-deoxyglucose labeling shows that normal
activity decreases in layers II/III and IV of barrel columns corresponding to plucked whiskers in
young adult rats. In rats deprived at older age, decreases in metabolic activity are reduced and only
measurable in layers II/III, and absent in layer IV (Kossut, 1998; Skibinska et al., 2000). Moreover,
plastic changes in responses from adult cortex after peripheral manipulations appear as early as 24
hrs later in layers II/III, but not in layer IV (Diamond et al., 1994). This induced changes occur in
layer II/III regardless of changes in layer IV after sensory manipulation and suggest that
supragranular layers are indeed a site of experience dependent plasticity in SI (Glazewski and Fox,
1996). Contribution to these changes might occur from layer IV to II/III synapses, since whisker
deprivation induces depression at these sites (Allen et al., 2003; Sheperd et al., 2003).
The present results show that GABAA receptor binding decreases not only in layer IV
(Fuchs and Salazar, 1998), but also in the deprived layers II/III of the same column. While layer IV
presents a high degree of functional independence from supragranular layers (Goldreich, et al.,
17
1999; Huang et al., 1998), intracortical activity is required for potentiation and depression of
sensory responses in barrel cortex (Wallace et al., 2001). Thus, a reduction in GABAA receptors in
layers II/III could significantly contribute to observed experience-dependent plasticity (Lendvai et
al., 2000; Stern et al., 2001; Fox, 1992, 1994; Simons and Land, 1987; Fox, 1992, 1994; Glazewski
and Fox, 1996; Wallace and Fox, 1999; Fuchs and Salazar, 1998). It would be interesting to test
whether infusing muscimol in the barrel cortex using an elvax implant has any effect in GABAA
receptors from deprived and intact cortical layers II/III and IV.
The decrease in GABAA receptors might have major functional consequences. For
instance, during development, deprivation causes axonal branches ascending from layer IV to
layers II/III to grow exubertantly and make errors, extending beyond their correspondent barrel
column (Stern et al., 2001). It seems that a decrease in GABAA receptors could allow the spreading
of excitation in layers II/III and thus affect the mechanisms that promote/inhibit axonal growth. In
the adult these effects would be less dramatic, once the synaptic connections are established.
Alpha1-GABAA receptor subunit immunocytochemistry
The present study is the first to show that chronic whisker trimming in adulthood decreases
the level of α1-GABAA receptor subunit staining in the corresponding columns. Reductions in α1GABAA receptor subunit have been reported in other cases of sensory deprivation. Monkeys
monocularly deprived with intravitreal injection of tetradotoxin for 8-21 days reduces levels of α1,
β2, and γ2 subunit mRNA in the deprived visual cortex. This reduction is fairly specific, since
mRNA for other subunits (α2, α4, and β1) remain unchanged (Huntsman et al., 1994; Jones, 1997).
Whether similar decreases in alpha-1 GABAA receptor occur after neonatal deprivation is not
known. Rat SI shows a decrease in α1 and α5-GABAA in focal cortical malformations induced by
18
neonatal freeze lesions of SI (Redecker, 2000). Furthermore, electrolytic lesion of thalamus in the
newborn decreases α1 in layers III-IV, but increases α2, α3, and α5 in the same areas (Paysan,
1997).
These changes in density of immunoreactive structures could either be a result of specific
down-regulation of receptor subunit, or represent a reduction in the inhibitory synaptic contacts, or
both. In adult monkeys, blockage of sensory input results in differential up- or down-regulation of
GABAA receptor genes (Hendry et al., 1994). Whether similar manipulations modify synaptic
contacts is addressed in the next section.
Nissl Results
In this study, adult whisker trimming results in significant decreases in all layers of
deprived barrel columns. This decrease could reflect either cell density changes, reduction of
Nissl-stained axons and dendrites, or shrinkage of the deprived columns (or expansion of the
undeprived, adjacent ones). Cell density changes have been reported after whisker deprivation.
GABA-containing neurons and GABAA receptors from deprived layer IV decrease in number after
whisker trimming (Micheva and Beaulieu, 1995b; Fuchs and Salazar, 1998). GABAergic
population in barrel cortex represents 10% of the total (Micheva and Beaulieu, 1995b).
Furthermore, neonatal all whisker-deprivation reduces the numerical density of presumed
inhibitory synapses by 52% (Sadaka et al., 2003). In contrast, when considering all barrel Nisslstained cells, neonatal whisker deprivation does not affect numerical density, but it significantly
decreases (13.7%) the intracortical projections in all layers of the deprived columns (Keller and
Carlson, 1999). This suggests that our results might represent a decrease in the Nissl-stained
projections rather than cell number change. The possibility of shrinkage of deprived barrel size is
19
not supported by studies of neonatal unilateral sensory deprivation (Sadaka et al., 2003). Our
results, however, were based on trimming only one row. This different way of depriving whiskers
might account for differences in functional and structural changes (Glazewski and Fox, 1996).
During development, whisker deprivation leads to abnormal receptive fields in layers II/III
(Stern et al., 2001). These abnormalities seem to be caused in part by aberrant ascending axons
from layer IV to layer II/III, by changes in inhibition, intrinsic membrane properties of cortical
neurons, and modulation of existing synapses by LTP. Some lines of evidence suggest that axonal
length within columns develops normally from layer IV to II/III even in absence of normal sensory
experience (Bender et al., 2003). However, comparisons between deprived and non-deprived barrel
columns show a decrease in connectivity in deprived neurons from layers II/III (Petersen et al.,
2004), which results from changes in decreased unitary EPSP amplitudes. Other deprivation
studies during critical periods show that dendritic branching in layer II/III neurons changes after
deprivation performed during critical periods only (Maravall et al., 2004).
Our results were based on deprived/nondeprived ratios. Against the possibility of axonal
increase in the adjacent-to-deprived row in layer II/III, is the finding that spine density decreased in
the deprived barrels. This reflects a decrease in synaptic density in layer III neurons, the likely site
of termination of cortico-cortical inputs, and indicated a decrease in functional synapses between
columns that may. In turn, this downregulation may account for the change in cortical
representation of the deprived row in these layers (Kossut, 1998).
20
Implications
Since sensory manipulation can induce changes in LII/III receptive fields before or without
it affecting LIV (Glazewski and Fox, 1996; Stern et al., 2001, the synapses from LIV to
LII/III have been hypothesized to be a site of experience-dependent plasticity in SI. Support
from Allen, et al, 2003, in which whisker deprivation induces synaptic depression at LIV to
L2/3 synapses. Also, Sheperd et, al., 2003 : it alters the functional topography of the L4 to
L2/3 projection.
Bender et al., 2003, on the other hand looked for the axonal changes from IIIV to II/III after
deprivation and found no change. Why do we find a change in Nissl? What does this
mean? Are there changes in Nissl reported for Layer IV?
Future experiments:
-
measure decrease in activity in infraorbital nerve after 1 week
-
compare neonatal vs adult deprivation for alpha 1, and in the neonatal should be
larger effect?
-
See if the effect is reversible in the neonatal and in the adult.
-
Compare C dperived versus ABDE deprived figures: is there an increase in the
C representation in the spare C in the abde clipped animal? For Alpha 1 or the
otheres? Kossut reports 65%increase after stimulation of spare C,in layer Iv in
2DGin surgery leaving intact C; no ipsilateral effects of stimulant. Increase
present in layer II-III and as well.
-
Measure width in micrometers of barrels of intact vs deprived?at different
laminar levels (see Kossut 98)
-
How was the significance calculated for II/III vs IV? was it by placing the
21
rqatios of decreases in normal and exp or by comparing ratios II/IIIover IV in
normals vs ratio II/III over IV in deprived? If so, then we got a winner!!! (The
mech for deprivation affecting IV is intracortical and what is affecting IV is not
necessary the same affecting II/III.
-
If decrease inn II/III is intracortical, then not only GABA, but also some
excitatory neurotransmitters might be affected in II/III even when not in IV
(NMDA?)(See discussion in Micheva ’95)
-
The decrease in alpha 1 GABAAR subunit can be caused either by decreased
synthesis of receptors or changes in subunit composition. (Lech , monica 2001)
-
Ask Dr. Fuchs if she has sections with deprived NMDA receptor binding!!
-
Point out that this study involves young adult (adolescent) rats, 6 weeks old.
Their brains might be more plastic than older adults (like older than 6 months,
i.e. Glazewski, and Fox, 1996)
-
Maybe mention the importance of these experiments in the determination of
mechanisms that can increase plasticity with therapeutic means, such as
decreasing gabaergic inhibition or increasing neuromodulator levels (Morales,
B., 2003)
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