Title: Effects of Whisker-trimming on GABA receptors in SI Cortex CHAPTER 1 INTRODUCTION

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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 spontaneous
activity, and the decrease in preference for an 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).There are two
suggested sites for origin of disinhibition: one thalamocortical and another cortical(
). It
seems that this increase disinhibition arises in cortex (Simons and Land, 1994), since deprivation
effects can be avoided by muscimol cortical blockage (Wallace et al., 2001). These cortical map
modifications seem to be compensatory, brought about by the elimination of part of sensory input
to the brain, as opposed to learning-dependent plasticity (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). Adult plasticity differs from
developmental plasticity in fundamental ways. In the adult, experience-regulated plasticity may be
a temporal, reversible event, and result of synapse formation and elimination (Trachtenberg et al.,
2000), or could only involve specific changes in synaptic strength (Rausell and jones 1995;
16
Huntley, 1996). These changes could be expected primarily in the 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). Other experiments support the
existence of critical periods.
Furthermore, whisker deprivation changes the excitability of neurons in cortex in a layer
specific manner. While layer IV becomes more sensitive to whisker deprivation around postnatal
day 7 (Fox, 1992), layers 2/3 and 5 receptive fields remain plastic (Armstrong-James et al., 1994;
Diamond et al., 1994; Glazewski and Fox, 1996; Lendvai et al, 2000, Sheperd et al, 2003). For
instance, 2DG labeling shows that normal activity decreases in layer IV of barrel columns
corresponding to plucked whiskers in adulthood (Kossut 1998). This downregulation in deprived
layer IV is even more extensive in deprived layers II/III in young adults. This is interpreted as a
loss of activation of ‘surround’ by neighboring barrels (Skibinska et al., 2000).
Consistent with these findings, 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 and III
of the same column. These observations suggest that the weaker responses observed in neurons
from layers II/III from deprived columns might be underlied by changes in GABAA receptors
(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). Previous studies show a non significant
decrease of GABA-containing somata synapses layer II and III of rat SI (Micheva and Beaulieu
1995).
What could explain the decrease in GABAergic elements observed in our study? Some
17
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).
In these exp we observe changes in All markers: GABAA, alpha 1 subunit and Nissl. Is the
difference caused by the amount of marker used (i.e. 1:100,000 units vs whatever they are using?
Micheva was suggesting that more important than dendritic or axonal changes might be the
changes at submicroscopic levels, and the receptor subunit changes might be sufficient to change
the efficiency () of the deal.
(The decreased disinhibition in barrel neurons thus could be an effect of the decline in
inhibitory neurotransmission mediated by GABAA receptors. Since the decrease in this study was
obtained through calculation of ratios, an alternative possibility could be that GABAA receptors in
the adjacent non-deprived rows could be actually increasing. However, there was no difference
between [3H]muscimol levels between rows adjacent and non-adjacent to a deprived row)
In Kossut, 1998, when comparing with controls, there was no increase of the spared
whisker, but a decrease in the deprived whiskers in spine density, which could reflect a decrease in
synaptic density in layer III neurons, which are likely site of termination of cortico-cortical inputs.
Kossut proposes this decrease as an indication for “weakening of functional links between the
columns and may underlie the observed downregulation of the cortical representation of the
deprived row of vibrissae”
18
Alpha1-GABAA receptor subunit immunocytochemistry
The present study shows that chronic whisker trimming in adulthood decreased the level of
α1-GABAA receptor subunit staining in the corresponding columns. Similar reductions in α1GABAA receptor subunit have been reported. Monocularly depived monkeys show reduced
levelsFor instance, monkeys monocularly deprived show reduced levels of α1, β2, and γ2 subunit
mRNA in the deprived visual cortex. This reduction is fairly specific, since α2, α4, and β1 subunit
mRNA levels remain unchanged (Huntsman et al., 1994; Jones, 1997). Also rat SI shows a
decrease in α1 and α5-GABAA in focal cortical malformations induced by 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 generic changes after deprivation….. When whiskers are trimmed during early
postnatal development, stimulation of the regrown whiskers causes a reduction in responses from
layer II/III neurons in the corresponding deprived column, whereas neighboring barrels columns
show stronger responses (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). In like manner, responses from layer II/III barrel
neurons are recorded in rats deprived as adults (Glazewski and Fox, 1996; Wallace and Fox, 1999).
However, the chemical basis for these changes inn layer II/III has yet to be determined.
Implications
1)
Since sensory manipulation can induce changes in LII/III receptive fields before
19
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 experiencedependent 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.
2)
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
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
20
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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