Cortical columns (barrels) display normal size in the brain’s primary somatosensory cortex of mice carrying null mutations of the insulin receptor substrate 1 gene: A preliminary report

doi:10.4236/abb.2013.410125

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Advances in Bioscience and Biotechnology, 2013, 4, 945-948 ABB

http://dx.doi.org/10.4236/abb.2013.410125 Published Online October 2013 (http://www.scirp.org/journal/abb/)

Cortical columns (barrels) display normal size in the

brain’s primary somatosensory cortex of mice carrying

null mutations of the insulin receptor substrate 1 gene:

A preliminary report

Marta López-Santibáñez Guevara1, Eileen Uribe-Querol2, Alma Lilia Fuentes Farías3*,

Esperanza Meléndez-Herrera3, Agustine Joseph D’Ercole4, Gabriel Gutiérrez-Ospina1,5*

1Departamento de Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de

México, México D.F., México

2División de Estudios de Posgrado e Investigación, Facultad de Odontología, Universidad Nacional Autónoma de México, México

D.F., México

3Departamento de Zoología, Instituto de Investigaciones sobre los Recursos Naturales, Universidad Michoacana de San Nicolás de

Hidalgo, Morelia, México

4Department of Pediatrics, The University of North Carolina at Chapel Hill, Chapel Hill, USA

5Coordinación de Psicofisiología, Facultad de Psicología, Universidad Nacional Autónoma de México, México D.F., México

Email: *[email protected], *[email protected]

Received 10 August 2013; revised 10 September 2013; accepted 25 September 2013

tribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is prop-

erly cited.

ABSTRACT

Circuits in barrels of the rodent brain’s primary

somatosensory (S1) cortex build up following con-

structivist rules. Previous evidence in mice supports

that the precise addition of barrel neuropil is pro-

moted by insulin-like growth factor-1 (IGF-1). The

signaling cascades mediating this response remain

undetermined. To address whether the effects of IGF-

1 upon the growth of S1 circuits are mediated by in-

sulin receptor substrate-1 (IRS-1), we studied barrel

size in adult mice having the IRS-1 gene knocked out

(IRS-1 ko). Our results reveal that barrel size is simi-

lar between wild type and IRS-1 ko mice suggesting

that IRS-1 is not essential for barrel circuitry growth.

Hence, investigations aimed at exploring other sub-

strates activated by IGF-1, namely IRS-2 and IRS-4,

are needed to reveal signaling pathways that mediate

the precise addition of S1 neuronal circuitry.

Keywords: Barrel Cortex; Neu ronal Cir cuit As semblage;

Trophic Interactions; Constructivism

1. INTRODUCTION

Conventional wisdom claims that mature neuronal cir-

cuits arise from an initially redundant set of connections

which are later refined through a process of selective

pruning driven by neuronal activity and competition

[1-4]. Empirical evidence suggests, however, that this

principle is far from universal since circuits in the spinal

cord [5,6], the olfactory bulb [7,8] and in the primary

visual [9] and somatosensory (S1) [10-13] cortices of

different mammals (however, see [14]), are predomi-

nantly elaborated by adding up connections and neuronal

processes [15].

The ce reb ral co rtex is for med by colu mns th at r un ver-

tically through its entire thickness. In S1, these columns

are called barrels and they represent collections of me-

chanosensory receptors distributed throughout the body.

In previous reports, we confirmed that barrel neuropil

and synaptic contacts are added through postnatal de-

velopment. More importantly, the ad dition of barrel neu-

ropil/synapses depended upon the availability of insulin-

like growth factor-1 (IGF-1). Indeed, barrels in trans-

genic mice overexpressing IGF-1 were enlarged, whereas

those in transgenic mice having brain ectopic expression

of IGF-binding-protein-1 displayed reduced size [16].

Both enlarged and small sized barrels had similar syn-

apse density [17]. These results thus support the concept

that neuronal circuits are elaborated by the precise, dif-

ferential addition of new elements through non-competi-

tive, constructive processes that depend upon the avail-

ability of neurotrophic factors (see [11] and [18] for a

*Corresponding authors.

OPEN ACCESS

M. L.-S. Guevara et al. / Advances in Bioscience and Biotechnology 4 (2013) 945-948

946

similar conclusion).

Considerable research has been conducted to under-

stand the cellular and molecular mechanisms that under-

lie the competitive and selective pruning of neuronal

connections. However, much less is known about the

molecular underpinnings supporting the formation and

growth of neuronal connections through constructivist

processes. Although molecules governing axonal and

dendritic navigation and growth may readily explain pre-

cise targeting and directed growth [19], the molecular

underpinnings that promote the precise addition of neu-

ronal circuits remain unexplored. Given that IGF-1 over-

expression promotes this process in S1 barrels, it is pos-

sible that molecules along the IGF-1 signaling pathway

may well underlie constructivist events during S1 as-

semblage.

IGF-1 binds to the IGF receptor typ e 1 that, after acti-

vation, phosphorylates signaling proteins of the insulin

receptor substrate (IRS) family. IRSs, in turn, activate

effector proteins containing Src homology 2 domains, a

process that activates downstream MAP Kinase and the

phosphatidylinositol 3-kinase (PI3K) signaling cascades

[20-23]. A member of the IRS family is IRS-1. IRS-1

mRNA is expressed in the cerebral cortex [24] and its

null mutation retards brain growth and reduces brain

weight, presumably by impairing IGF-1 actions on brain

growth [25,26]. IRS-1 might then be involved in promot-

ing the precise and progressive addition of barrel neu-

ropil following IGF-1 binding to its receptor. Hence, by

combining succinic dehydrogenase (SDH) histochemis-

try and image analyses, we evaluated whether adult wild

type (wt) mice and those carrying null mutations of the

IRS-1 gene (IRS-1ko mice) displayed differences in bar-

rel size.

2. MATERIALS AND METHODS

2.1. Mice

Studies were performed in genotyped adult male wild

type (n = 3) and homozygous IRS-1 ko mice (n = 4) gen-

erously provided by Dr. C. Ronald Kahn (Joslin Diabetes

Center and Department of Medicine, Harvard Medical

School, Boston, MA). Upon arrival, mice were kept until

the day of sacrifice under 12:12 hours light/dark cycle at

22˚C having free access to food and water. Animal han-

dling and procedures followed the guidelines of the NIH

guide for the care and use of experimental animals and

were approved by the institutional review committee at

the University of North Carolina (UNC), Chapel Hill.

2.2. Histological Procedures

The anesthetized (ketamine/xylazine; 900/20 µg/gr body

weight) mice were perfused with physiological saline

followed by 10% glycerol. Brains were removed and

weighed and the cerebral cortex was dissected and frozen

flattened [13]. Serial tangential sections (30 µm) were

cut in a cryostat and stained for the activity o f the oxida-

tive enzyme, succinic dehydrogenase (SDH) by means of

SDH histochemistry [13]. Briefly, sections were dried on

the slides at 37˚C for 30 min; fixed in 10% formalin, pH

7.6, at 4˚C for 1 min and rinsed in distilled water. Then

they were stained by reacting them with 0.05 M sodium

succinate and 0.55 mM nitroblue tetrazolium in 0.05 M

phosphate buffer, pH 7.6. Slides were rinsed and cover-

slipped with DPX. Animal perfusion and sample staining

were carried out at UNC.

2.3. Estimation of Barrel Cross-Sectional and

Posteromedial Barrel Subfield Areas

As previously commented, S1 barrels in the mouse brain

represent collections of mechanosensory receptors lo-

cated along the body. The body has different segments

though. Barrels are then segregated into distinct body

representations. From these representations, the most

conspicuous is that known as the posteromedial barrel

subfield (PMBSF) in which each PMBSF barrel repre-

sents each facial vibrissae. The histological material ob-

tained from wt and IRS-1 ko mice was transferred to the

Universidad Nacional Autónoma de México where mor-

phometry of PMBSF barrels was conducted. Two dimen-

sional maps of PMBSF were drawn under bright field

microscope equipped with a camera lucida. Completed

maps were digitized and PMBSF and barrel cross-sec-

tional areas were estimated using Image-Pro (Media Cy-

bernetics, Silver Spring, MD), as described elsewhere

[13].

2.4. Data Analyses

Data are reported as means ± standard deviation for both

experimental groups. PMBSF barrel area and barrel

cross-sectional area were statistically compared between

wt and IRS-1 ko mice by using One-way ANOVA test

followed by a Tukey’s post hoc test (OriginPro v 7;

OriginLab Corporation) setting the significance value at

p < 0.05.

3. RESULTS

The number, shape and anatomical definition of the

PMBSF were fully comparable between wt and IR S-1 ko

mice (Figure 1). Although average barrel area (wt: 0.04761

± 0.00256 versus IRS-1 ko: 0.04481 ± 0.00076 both in

cm2), total barrel field area (wt: 1.5233 ± 0.08172 versus

IRS-1 ko: 1.4673 ± 0.03872 both in cm2) and PMBSF

area (wt: 1.8645 ± 0.16628 versus IRS-1 ko: 1.6626 ±

0.04809 both in cm2) tended to be reduced in IRS-1 ko

mice, such trends were not statistically significant.

M. L.-S. Guevara et al. / Advances in Bioscience and Biotechnology 4 (2013) 945-948 947

wt IRS-1 ko

Figure 1. Representative camera lucida drawings of the barrel

field of wild type (wt) and IRS-1 knock out (IRS-1 ko) mice.

No qualitative differences were observed between mouse groups

(Scale = 1 mm).

4. DISCUSSION

Previous reports in rodents support that barrel circuitry in

S1 builds up following constructivist principles [10-13,

15]. We have shown that the precise and progressive ad-

dition of barrel neuropil is promoted by IGF-1 [15,16].

The signaling cascade involved in this event has not been

elucidated. We then explored the contribution of IRS-1 in

this process by comparing the adult size of SI barrels

between wt and IRS-1 ko mice. IRS-1 has been previ-

ously shown to be expressed in the cerebral cortex [24]

and its mutation retards brain growth and reduces brain

weight [25,26]. Unexpectedly, IRS-1 ko mice displayed

barrel and PMBSF areas fully comparable to those ob-

served in wt mice, thus supporting that IRS-1 is not es-

sential for promoting the precise addition of barrel neu-

ropil. In a previous report, Ye et al. [26] have shown that

IRS-2 and IRS-4 may compensate the lack of IRS-1 with

regard to myelination processes, and it is therefore con-

ceivable that either one or both could also “rescue” the

barrel’s phenotype in IRS-1 ko mice. In sum, IRS-1 does

not seem to mediate the trophic effects of IGF1 on the

barrel cortex. Future experiments must address whether

IRS-2 and/or IRS-4 participate in the construction of

barrel neuropil.

5. ACKNOWLEDGEMENTS

Authors thank Jesús Ramirez Santos, Ivonne Mora, Edel Pineda Lopez

and Raymundo Reyes for technical assistance. This work was sup-

ported in part by CONACyT (Grant No. 82879 to G. G. O.), PAPIIT-

UNAM (Grants Nos. IN203912-3 to G. G. O. and IA202013-2 to E. U.

Q.), CIC-UMSNH (Grant No. 8.37 to A. L. F. F.).

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