Sexually monomorphic maps and dimorphic responses in rat genital cortex

Author Posting. © The Author(s), 2015. This is the author's version of the work. It is posted here for personal use, not for redistribution. The definitive version was published in Current Biology 26 (2016): 106-113, doi:10.1016/j.cub.2015.11.041.Mammalian external genitals show sexual dimorphism [1,2] and can change size and shape upon sexual arousal. Genitals feature prominently in the oldest pieces of figural art [3] and phallic depictions of penises informed psychoanalytic thought about sexuality [4, 5]. Despite this longstanding interest, the neural representations of genitals are still poorly understood [6]. In somatosensory cortex specifically, many studies did not detect any cortical representation of genitals [7-9]. Studies in humans debate, if genitals are represented displaced below the foot of the cortical body map [10-12], or if they are represented somatotopically [13-15]. We wondered, what a high-resolution mapping of genital representations might tell us about the sexual differentiation of the mammalian brain. We identified genital responses in rat somatosensory cortex in a region previously assigned as arm/leg cortex. Genital responses were more common in males than in females. Despite such response dimorphism, we observed a stunning anatomical monomorphism of cortical penis and clitoris input maps revealed by cytochrome-oxidasestaining of cortical layer-4. Genital representations were somatotopic, bilaterally symmetric and their relative size increased markedly during puberty. Size, shape and erect posture give the cortical penis representation a phallic appearance pointing to a role in sexually aroused states. Cortical genital neurons showed unusual multi-body-part responses and sexually dimorphic receptive fields. Specifically, genital neurons were coactivated by distant body regions, which are touched during mounting in the respective sex. Genital maps indicate a deep homology of penis and clitoris representations in line with a fundamentally bi-sexual layout [16] of the vertebrate brain.This work was supported by Marine Biological Laboratory, Humboldt Universität zu Berlin and Neurocure. M.B. was a recipient of a Gottfried Wilhelm Leibniz Prize

Pax6 interactions with chromatin and identification of its novel direct target genes in lens and forebrain.

Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of α-, β- and δ-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip) were performed using three distinct sources of chromatin (lens, forebrain and β-cells). ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133) of these promoter regions were shared between at least two (three) distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6⁺/⁻ lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6⁻/⁻ lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6 interacts preferentially with promoter regions in a tissue-specific fashion. Nevertheless, nearly 20% of the regions identified are accessible to Pax6 in multiple tissues

Aberrant cerebellar Purkinje cell activity as the cause of motor attacks in a mouse model of episodic ataxia type 2

Many cerebellar-induced neurological disorders, such as ataxias and cerebellar-induced dystonias, are associated with abnormal Purkinje cell activity. In tottering mice, a well-established mouse model of episodic ataxia type 2 (EA2), cerebellar Purkinje cells are required for the initiation of motor attacks. How Purkinje cells contribute to the initiation of attacks is not known, and to date there are no reports on the activity of Purkinje cells during motor attacks in the tottering mice. Here, we show that tottering Purkinje cells exhibit high-frequency burst firing during attacks, reminiscent of other mouse models of cerebellar-induced motor dysfunction. We recorded the activity of Purkinje cells in awake head-restrained tottering mice at baseline, or during caffeine-induced attacks. During motor attacks, firing of Purkinje cells transformed to high-frequency burst firing. Interestingly, the extent to which the activity of Purkinje cells was erratic was correlated with the severity of the motor dysfunction. In support of a causal role for erratic activity in generating motor dysfunction, we found that direct infusion of the small conductance calcium-activated potassium (SK) channel activator NS309 into the cerebellum of tottering mice in the midst of an attack normalized the firing of Purkinje cells and aborted attacks. Conversely, we found that inducing high-frequency burst firing of Purkinje cells in wild-type animals is sufficient to produce severe motor signs. We report that erratic activity of wild-type Purkinje cells results in ataxia and dystonic postures. Moreover, this aberrant activity is the cause of motor attacks in the tottering mice

Hypothermia-induced dystonia and abnormal cerebellar activity in a mouse model with a single disease-mutation in the sodium-potassium pump

<div><p>Mutations in the neuron-specific α₃ isoform of the Na⁺/K⁺-ATPase are found in patients suffering from Rapid onset Dystonia Parkinsonism and Alternating Hemiplegia of Childhood, two closely related movement disorders. We show that mice harboring a heterozygous hot spot disease mutation, D801Y (α₃^+/D801Y), suffer abrupt hypothermia-induced dystonia identified by electromyographic recordings. Single-neuron <i>in vivo</i> recordings in awake α₃^+/D801Y mice revealed irregular firing of Purkinje cells and their synaptic targets, the deep cerebellar nuclei neurons, which was further exacerbated during dystonia and evolved into abnormal high-frequency burst-like firing. Biophysically, we show that the D-to-Y mutation abolished pump-mediated Na⁺/K⁺ exchange, but allowed the pumps to bind Na⁺ and become phosphorylated. These findings implicate aberrant cerebellar activity in α₃ isoform-related dystonia and add to the functional understanding of the scarce and severe mutations in the α₃ isoform Na⁺/K⁺-ATPase.</p></div

Dystonia. Hypothermia-induced attacks are dystonic of nature.

<p>(A) Illustration showing the locations of the ECoG electrodes. ECoG was bilaterally recorded from the primary motor cortex with ground and reference electrodes placed above the superior colliculi. (B) Picture of the experimental setting showing a α₃^+/D801Y mouse freely moving in an empty cage while ECoG is recorded. (C) Representative example of ECoG (left) and corresponding power spectrum of a baseline measurement during which the mouse is exploring the cage. (D) As in C but the recording was made during an attack induced by cold water exposure in the same α₃^+/D801Y mouse. (E) As in C and D but recorded during a pilocarpine induced tonic-clonic seizure in the same mouse (note the difference in y-axis of both the ECoG and power spectrum). (F) Illustration indicating locations of EMG recordings from the tibialis and gastrocnemius in the hind limb. (G, H) Representative examples of EMG recorded from the same α₃^+/D801Y mouse from the anterior tibialis and gastrocnemius pre (B, blue) and post (C, green) a cold water induced attack. (I) Cross correlograms of the traces shown in G (blue) and H (green) showing a pronounced difference in correlation between activity of agonist and antagonist hind limb muscles indicative of dystonic postures during an attack.</p

Ataxia. α₃^+/D801Y mice display moderate motor deficits.

<p>(A) Gait analysis with fore and hind base width and stride length (n = 6 for both WT and α₃^+/D801Y). Front paws were colored blue, while hind paws were colored with red paint. (B) Hind limb clasping test (n = 10 for WT and n = 6 for α₃^+/D801Y). (C) Balance beam test over 3 consecutive days, with time to cross (left) and number of slips (right) (n = 24 for WT and n = 23 for α₃^+/D801Y). (D) Rope climb test with time to climb (n = 19 for WT and n = 23 α₃^+/D801Y). (E) Parallel rod floor test with distance traveled, number of slips and ataxia ratio defined by: number of slips/(distance*100) (n = 10 for WT and n = 12 for α₃^+/D801Y mice). (F) Grip strength (n = 12 for WT and n = 13 for α₃^+/D801Y). All data shown are means ± SEM. *p<0.05, **p<0.01, ***p<0.001.</p

Hypothermic attacks. Hypothermia causes dystonia-like attacks in α₃^+/D801Y mice.

<p>(A) Average occurrence (%) of an attack in α₃^+/D801Y mice, following restraining for 10 min (n = 5), tail suspension for 6 min (n = 6), randomly timed electric foot shocks (n = 5), exposure to fox urine (n = 5), warm incubator (43°C) (n = 5), temperate water swim (35°C) (n = 6), chronic variable stress protocol (n = 11), cold water swim (5–10°C) (n = 10), cold environment (-20°C) (n = 6) and Prazosin treatment before cold water swim (n = 5). Only hypothermia, caused by cold water swim or cold environment exposure, consistently induced attacks in the α₃^+/D801Y mice (n = 15 for cold water and n = 6 for cold environment). (B) Example of dystonic-like posture with hind limbs hyperextended caudally (left picture, arrow) and a period of convulsion with abnormal postures and twisting movements (right picture) in α₃^+/D801Y mice after cold water swim. WT mice never displayed similar abnormal symptoms (left picture). (C) Core body temperature measured by rectal probe at onset of attack induced by exposure to cold water or cold environment. Both methods induced a significant drop in body temperature just below about 20°C before symptoms occurred in α₃^+/D801Y mice. WT mice displayed identical drops in body temperature (n = 6 for both WT and α₃^+/D801Y). (D) Attack duration after induction by cold water when α₃^+/D801Y mice were left to recuperate at room temperature or on a 33.3°C heating pad (n = 6).</p

Cerebellar activity. <i>In vivo</i> recordings of awake α₃^+/D801Y mice revealed irregular firing of Purkinje cells and DCN neurons, which during dystonic spells was further exacerbated and turned into periods of abnormal high-frequency bursting.

<p>(A) Illustration of an <i>in vivo</i> recording of Purkinje cells in awake head-restrained mice. (B) Representative raw traces of Purkinje cells recorded from WT, α₃^+/D801Y at baseline, and α₃^+/D801Y mice during dystonic attack induced by cold water. Scale bars: 500 ms by 50 μV. (C) Average firing rate (upper), predominant firing rate (middle) and CV ISI (lower) of Purkinje cells from WT (N = 4 (animals), n = 19 (cells)), α₃^+/D801Y at baseline (N = 5, n = 23), control WT exposed to cold water (N = 3, n = 18) and α₃^+/D801Y mice during dystonic attacks induced by cold water (N = 4, n = 20). (D) Illustration of an <i>in vivo</i> recording of DCN neurons in awake head-restrained mice. (E) Representative raw traces of DCN neurons recorded from WT, α₃^+/D801Y at baseline, and α₃^+/D801Y mice during dystonic attack induced by cold water. (F) Average firing rate (upper), predominant firing rate (middle) and CV ISI (lower) of DCN neurons from WT (N = 4 (animals), n = 21 (cells)), α₃^+/D801Y at baseline (N = 5, n = 21), control WT mice exposed to cold water (N = 3, n = 18) and α₃^+/D801Y mice during dystonic attacks induced by cold water (N = 4, n = 20). All data shown are means ± SEM. *p<0.05, **p<0.01, ***p<0.001.</p

<i>In vitro</i> pump function. Functional assays of Na⁺/K⁺ ATPases with substitutions in the disease hotspot aspartate residue.

<p>(A, B, C) Currents recorded in Na⁺-loaded oocytes expressing exogenous ouabain-resistant Na⁺/K⁺-ATPases without (wild type, A), or with, a D-to-Y (B) or D-to-N (C) mutation at position 801 equivalent, held at -20 mV, exposed to 125 mM Na⁺ solution at pH 7.6 containing 1 μM ouabain (to silence endogenous Na⁺/K⁺-ATPases), with 15 mM K⁺ added as indicated by horizontal bars (Ko); the vertical lines are responses to 50-ms steps to other potentials. (D, E, F) Steady-state current levels plotted against voltage, from the recordings shown in (A, B, C) (filled symbols), in the presence (red) or absence (black) of K⁺, and from subsequent recordings in the same oocyte after inhibition of exogenously expressed pumps by 10 mM ouabain (empty symbols). (G, H, I) Average ± SEM 10 mM ouabain-sensitive steady currents (I ouab-sens) in 125 mM Na⁺, obtained by subtraction, at 0 mM K⁺ (black circle) or 15 mM K⁺ (red triangle), normalized to the maximum Na⁺ charge movement in each oocyte (J-O, below), a measure of the number of Na⁺/K⁺-ATPases; wild type (n = 4 oocytes), D-to-Y (n = 3 with K⁺, n = 6 without), D-to-N (n = 3). (J, K, L) 10 mM ouabain-sensitive pre-steady-state Na⁺ currents for wild type (J), D-to-Y (K), and D-to-N (L) Na⁺/K⁺-ATPases in 125 mM Na⁺ and 0 mM K⁺ solution obtained by subtraction of traces before and after pump inhibition; superimposed traces are from steps to voltages between -180 mV and +60 mV, and back to the holding potential, -20 mV. (M, N, O) Transient Na⁺ charge movements, ΔQ, obtained as the time integral of the transient currents at -20 mV after each voltage step, are plotted against potential during the step for wild type (M), D-to-Y (N), and D-to-N (O) Na⁺/K⁺-ATPases. Boltzmann relation fits to the ΔQ-V plots yielded maximum ΔQ values used for normalization (ΔQ norm), and mean fit values for effective valence, zq (wild type: 0.68±0.01, n = 9; D-to-Y: 0.38±0.02, n = 6; D-to-N: 0.48±0.02, n = 9), and for midpoint voltage (wild type: -24±1 mV, n = 9; D-to-Y: -51±3 mV, n = 6; D-to-N: -19±2 mV, n = 9); maximum ΔQ for D-to-Y pumps is likely underestimated due to the lower zq, so that D-to-Y currents normalized to maximum charge (H, above) may be overestimated; averaged ΔQ norm-V distributions are shown. See also Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006763#pgen.1006763.s002" target="_blank">S2 Fig</a>.</p