Cellular structure and sexual development of somatosensory cortex

Schaltkreise der Großhirnrinde von Säugetieren bestehen aus unterschiedlichen Zellarten, deren charakteristische Physiologie, Morphologie und Konnektivität die Verarbeitung eintreffender neuronaler Signale bestimmen. Im Rahmen dieser Doktorarbeit wurde die zelluläre Spezialisierung zweier kortikaler Schaltkreise untersucht: einerseits die der Eingangsschicht oder Schicht 4 (Kapitel 2 bis 4) und andererseits die der Ausgangsschicht oder Schicht 5 (Kapitel 5). Der erste Teil der Dissertation umfasst drei Publikationen, welche die Verarbeitung von genitalen Berührungen in der kortikalen Schicht 4 von Nagern als Forschungsgegenstand haben. Die Arbeiten konzentrieren sich auf strukturelle Veränderungen während der Pubertät, da sich die Bedeutung genitaler Berührungen in dieser Zeit stark verändert. Die Erforschung des „Barrel Cortex“ führte zu der Erkenntnis, dass der somatosensorische Kortex eine topographische Repräsentation der Körperoberfläche enthält. Diese wird kurz nach der Geburt gebildet und weist danach kaum Veränderungen auf. Erstaunlicherweise vergrößert sich während der Pubertät der Bereich dieser Körperkarte, der genitale Berührungen verarbeitet. Dieser Prozess kann durch frühe sexuelle Berührungen beschleunigt werden. Im zweiten Teil dieser Dissertation wurde die kortikale Ausgangsstruktur untersucht. Diese ist von verschiedenen Projektionsneuronen besiedelt. Angesichts der drastischen Größenunterschiede dieser Projektionsneurone haben wir deren Genomgröße untersucht. Unsere Ergebnisse legen nahe, dass einige außerordentlich große Projektionsneurone zusätzliche Kopien ihres gesamten Chromosomensatzes enthalten. Insgesamt wurden in dieser Dissertation zwei neue Formen zellulärer Spezialisierung in der Hirnrinde aufgezeigt: (i) Schicht 4 weist zelltypspezifische entwicklungs- und erfahrungsabhängige Veränderungen im Genitalfeld auf. (ii) Schicht 5 enthält Projektionsneurone, deren erstaunliche Zellgröße auf ein polyploides Genom zurückzuführen ist.Functionally specialized circuits in the mammalian neocortex contain different neuronal cell-types, which process information depending on their physiology, morphology and synaptic connectivity. This doctoral thesis explores two functionally distinct cortical circuits, namely its input (Chapters 2 to 4) and output (Chapter 5) structures, layer 4 and layer 5 respectively. The first part of the thesis comprises three studies that examine the processing of genital touch in the cortical input layer, layer 4. We investigated how layer 4 and its inputs are structurally refined during puberty, a time when genital touch gains biological relevance. Earlier work from layer 4 of barrel cortex suggested that somatosensory cortex contains a topographic representation of the body surface. We find that the part of this body map that processes genital touch expands significantly during puberty and that this expansion could be advanced by the early experience of sexual touch. Our data further suggests that this expansion is not due to differences in the peripheral innervation of genitals. Finally, chronic imaging of excitatory neurons within layer 4 revealed cell-type specific functional and structural changes within genital cortex during puberty. The second part of this thesis focuses on the cellular specialization of the cortical output layer, layer 5, which contains different types of excitatory projection neurons. We investigated genomic differences as novel mechanism underlying projection neuron diversity. Our data suggests that some exceptionally large projection neurons may contain an increased DNA content, a phenomenon also referred to as polyploidy. Overall, this thesis highlights two novel instances of cellular specialization in the cortex: (i) Within the cortical input layer, we observed development and experience driven changes in the area which processes genital touch. (ii) Within the cortical output layer, we identified putatively polyploid projection neurons

Effects of Sexual Experience and Puberty on Mouse Genital Cortex revealed by Chronic Imaging

The topographic map in layer 4 of somatosensory cortex is usually specified early postnatally and stable thereafter. Genital cortex, however, undergoes a sex-hormone- and sexual-touch-dependent pubertal expansion. Here, we image pubertal development of genital cortex in Scnn1a-Tg3-Cre mice, where transgene expression has been shown to be restricted to layer 4 neurons with primary sensory cortex identity. Interestingly, during puberty, the number of Scnn1a+ neurons roughly doubled within genital cortex. The increase of Scnn1a+ neurons was gradual and rapidly advanced by initial sexual experience. Neurons that gained Scnn1a expression comprised stellate and pyramidal neurons in layer 4. Unlike during neonatal development, pyramids did not retract their apical dendrites during puberty. Calcium imaging revealed stronger genital-touch responses in Scnn1a+ neurons in males versus females and a developmental increase in responsiveness in females. The first sexual interaction is a unique physical experience that often creates long-lasting memories. We suggest such experience uniquely alters somatosensory body maps.Peer Reviewe

Constant innervation despite pubertal growth of the mouse penis

The sexual characteristics of the vertebrate body change under the control of sex hormones. In mammals, genitals undergo major changes in puberty. While such bodily changes have been well documented, the associated changes in the nervous system are poorly understood. To address this issue, we studied the growth and innervation of the mouse penis throughout puberty. To this end, we measured length and thickness of the mouse penis in prepubertal (3–4 week old) and adult (8–10 week old) mice and performed fiber counts of the dorsal penile nerve. We obtained such counts with confocal imaging of proximal sections of the mouse penis after paraffin embedding and antibody staining against Protein-Gene-Product-9.5 or Neurofilament-H in combination with antigen retrieval procedures. We find that the mouse penis grows roughly 1.4 times in both thickness and length. Fiber counts in the dorsal penile nerve were not different in prepubertal (1,620 ± 165 fibers per penis) and adult (1,572 ± 383 fibers per penis) mice, however. Antibody staining along with myelin staining by Luxol-Fast-Blue suggested about 57% of penile nerve fibers were myelinated. Quantification of the area of mouse somatosensory penis cortex allowed us to compare cortical magnification of the penile cortex and the whisker-barrel-cortex systems. This comparison suggested that 2 to 4 times less cortical area is devoted to a penile-nerve-fiber than to a whisker-nerve-fiber. The constant innervation of mouse penis through puberty suggests that the pubertal increase of cortical magnification of the penis is not simply a reflection of peripheral change.Peer Reviewe

Peeking into the sleeping brain: Using in vivo imaging in rodents to understand the relationship between sleep and cognition

Sleep is well known to benefit cognitive function. In particular, sleep has been shown to enhance learning and memory in both humans and animals. While the underlying mechanisms are not fully understood, it has been suggested that brain activity during sleep modulates neuronal communication through synaptic plasticity. These insights were mostly gained using electrophysiology to monitor ongoing large scale and single cell activity. While these efforts were instrumental in the characterisation of important network and cellular activity during sleep, several aspects underlying cognition are beyond the reach of this technology. Neuronal circuit activity is dynamically regulated via the precise interaction of different neuronal and non-neuronal cell types and relies on subtle modifications of individual synapses. In contrast to established electrophysiological approaches, recent advances in imaging techniques, mainly applied in rodents, provide unprecedented access to these aspects of neuronal function in vivo. In this review, we describe various techniques currently available for in vivo brain imaging, from single synapse to large scale network activity. We discuss the advantages and limitations of these approaches in the context of sleep research and describe which particular aspects related to cognition lend themselves to this kind of investigation. Finally, we review the few studies that used in vivo imaging in rodents to investigate the sleeping brain and discuss how the results have already significantly contributed to a better understanding on the complex relation between sleep and plasticity across development and adulthood

Development of rat female genital cortex and control of female puberty by sexual touch

<div><p>Rat somatosensory cortex contains a large sexually monomorphic genital representation. Genital cortex undergoes an unusual 2-fold expansion during puberty. Here, we investigate genital cortex development and female rat sexual maturation. Ovariectomies and estradiol injections suggested sex hormones cause the pubertal genital cortex expansion but not its maintenance at adult size. Genital cortex expanded by thalamic afferents invading surrounding dysgranular cortex. Genital touch was a dominant factor driving female sexual maturation. Raising female rats in contact with adult males promoted genital cortex expansion, whereas contact to adult females or nontactile (audio-visual-olfactory) male cues did not. Genital touch imposed by human experimenters powerfully advanced female genital cortex development and sexual maturation. Long-term blocking of genital cortex by tetrodotoxin in pubescent females housed with males prevented genital cortex expansion and decelerated vaginal opening. Sex hormones, sexual experience, and neural activity shape genital cortex, which contributes to the puberty promoting effects of sexual touch.</p></div

Artificial genital touch drives genital cortex expansion and promotes female sexual maturation.

<p>(A) Control, prepubescent female rats (postnatal day [P]23) were placed each day 3 times for 10 minutes in a box across 7 days without further treatment. (B) Artificial genital touch; prepubescent female rats (P23) were placed each day 3 times for 10 minutes in a box across for 7 days, and their clitoris and vulva were stroked with a lubricated brush by a human (female) experimenter. (C) Outline of a somatosensory cortex (S1) map from the brain of a P30 female who received the control treatment described in A. Genital cortex is depicted in black. (D) Same as (C), but example map stems from the brain of a P30 female, whose genitals were brushed as described in (B). (E), Absolute area of clitoris representation in hemispheres of P30 females, which underwent control (A) or artificial genital touch (B) treatment. (F) Same as (E), but the fraction of genital cortex of the S1 is plotted. (G) Same as (E), but the absolute area of S1 is shown for the 2 groups. There is no difference in S1 size. (H) Mean scores for vaginal opening for P30 females, which underwent control (A) or artificial genital touch (B) treatment. (I) Picture showing the uterus of a female, which underwent control (A) or artificial genital touch (B) treatment. (J) Uterine weights of females that underwent control (A) or artificial genital touch (B) treatment. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001283#pbio.2001283.s007" target="_blank">S1 Data</a>.</p

Systemic estradiol application drives genital cortex growth and advances puberty.

<p>(A) Upper panel: Control rats received daily subcutaneous injection of sesame oil for 5 days (postnatal day [P]26–P30). Lower panel: Outline of a somatosensory cortex (S1) map from the brain of a P30 female treated only with oil. The genital cortex is labeled in black. (B) Same as (A), but prepubescent rats were injected with sesame oil containing estradiol. (C) Absolute area of the clitoris representation in hemispheres of P30 females, which received daily injections (over 5 days) of either sesame oil alone or sesame oil containing estradiol. The genital cortex appears bigger in the estradiol group. (D) Same as (C), but the fraction of genital cortex of the entire S1 is plotted. (E) Absolute area of S1 in hemispheres of animals that received either daily oil or estradiol injections. There is no difference between the 2 groups. (F) Pictures of clitoris and vagina, before and after sesame oil treatment. Note that the vagina stays closed at P30 when animals were injected with sesame oil alone. (G) Same as (F), but for the vagina of estradiol treated animals. The vagina is already open at P30 (lower picture). (H) Mean scores for vaginal opening. A score of 0 represents a closed vagina, whereas a score of 1 stands for an opened vagina. The intermediate state (between open and closed vagina) has a score of 0.5. The vagina stayed closed in almost all control animals (treated with oil) whereas the majority of estradiol animals, showed an open vagina at P30. Each dot represents the score of vaginal opening for one animal. (I) Uterus weights of oil and estradiol treated animals. The uterus in estradiol treated animals is heavier, although this difference was not significant. Each dot represents the uterus weight from one animal. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001283#pbio.2001283.s003" target="_blank">S3 Fig</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001283#pbio.2001283.s007" target="_blank">S1 Data</a>.</p

Genital cortex growth in prepubescent females is accelerated by tactile cues from males.

<p>(A) Prepubescent animals (postnatal day [P]21) were cohoused for 9 days with either an adult, sexually experienced female (upper panel) or a sexually experienced adult male (middle panel). Whereas tactile contact was allowed in both of these groups, the third group of prepubescent rats was only exposed to olfactory (by a daily exchange of bedding between the cage compartments), visual, and auditory cues of a sexually experienced adult male (lower panel). (B) Upper panel: Outline of a somatosensory cortex (S1) map from the brain of a P30 female cohoused with a sexually experienced adult female. Genital cortex is labeled in black. Middle panel: Map from a brain of a P30 female cohoused with a sexually experienced adult male. Lower panel: Outline of a S1 map obtained from a P30 female exposed to olfactory, visual, and auditory cues but not tactile cues from a sexually experienced adult male. (C) Absolute area of genital cortex in hemispheres of P30 females cohoused with either a female (female and contact), a male (male and contact), or with a male without having tactile contact (male and no contact). Note that the size of genital cortex in the brains of animals who were cohoused in tactile contact with a male is significantly larger compared to the other 2 groups (female and contact, and male and no contact). (D) Same as (C), but the fraction of genital cortex of the entire S1 is shown. (E) Same as (C), but the absolute area of S1 is plotted. There is no difference between the 3 groups. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001283#pbio.2001283.s007" target="_blank">S1 Data</a>.</p

Pubertal expansion of genital cortex, but not its maintenance in adults requires sex hormones.

<p>(A) Outline of a somatosensory cortex (S1) map obtained from a female, aged postnatal day (P)25. Genital cortex is labeled in black. (B) Same as (A), but for an adult female (P42). Note the remarkable size difference of the genital cortex compared to the P25 female. (C) Outline of a S1 map from the brain of an adult female (P42), in which the ovaries were removed at P20. (D) Same as (B), but for an adult female (P60), in which the ovaries were removed at P42. The area of the genital cortex is similar to a nontreated adult female (B) and is bigger than in female rats ovariectomized before puberty (C). (E) Absolute area of clitoris in hemispheres of P25, P42 females, and females which were ovariectomized at either P20 or P42. (F) Fraction of genital cortex of the entire S1 in hemispheres of P25, P42 females, and females which were ovariectomized at either P20 or P42. Note that there is a substantial growth of the genital cortex between P25 and P42 animals. Female rats ovariectomized during prepuberty had smaller genital cortices than animals ovariectomized after puberty. (G) Absolute area of S1 in hemispheres of P25, P42, in prepuberty (P20) ovariectomized, and postpuberty (P42) ovariectomized female rats. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001283#pbio.2001283.s001" target="_blank">S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001283#pbio.2001283.s002" target="_blank">S2</a> Figs, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001283#pbio.2001283.s006" target="_blank">S1 Table</a>, and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001283#pbio.2001283.s007" target="_blank">S1 Data</a>.</p

Genital cortex growth is due to the invasion of dysgranular territories.

<p>(A) Upper panel: Map of somatosensory cortex (S1) obtained from a female aged postnatal day (P) 25. Genital cortex is labeled in black. The interlimb cortex is marked in grey. The upper red lines marks the forepaw axis. The lower red line is rectangular to the forepaw axis, terminates at the anterior end of the hindpaw representation, and was used to demarcate interlimb cortex. Lower panel: Corresponding for cytchrome c stained tangential section, which shows best the clitoris area. (B) Same as (A), but for an animal aged P48. Note that the interlimb cortex area is smaller than in the young animal and that the clitoris representation is larger. (C) Absolute area of the clitoris representation in hemispheres of young (P25) and old (P42) animals. The clitoris representation almost doubles in size. (D) Same as (C), but the absolute area of the interlimb cortex is plotted. The interlimb cortex is slightly smaller in area in old animals than in young animals. (E) The ratio of the genital cortex to the interlimb cortex increases after puberty in P42 animals. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001283#pbio.2001283.s004" target="_blank">S4 Fig</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001283#pbio.2001283.s007" target="_blank">S1 Data</a>.</p