Neurons in the developing CNS tend to send out long axon collaterals to multiple target areas. For these neurons to attain specific connections, some of their axon collaterals are subsequently pruned-a process called stereotyped axon pruning. One of the most striking examples of stereotyped pruning in the CNS is the pruning of corticospinal tract (CST) axons. The long CST collaterals from layer V neurons of the visual and motor cortices are differentially pruned during development. Here we demonstrate that select plexins and neuropilins, which serve as coreceptors for semaphorins, are expressed in visual cortical neurons at the time when CST axon collaterals are stereotypically pruned. By analyzing mutant mice, we find that the pruning of visual, but not motor, CST axon collaterals depends on plexin-A3, plexin-A4, and neuropilin-2. Expression pattern study suggests that Sema3F is a candidate local cue for the pruning of visual CST axons. Using electron microscopic analysis, we also show that visual CST axon collaterals form synaptic contacts in the spinal cord before pruning and that the unpruned collaterals in adult mutant mice are unmyelinated and maintain their synaptic contacts. Our results indicate that the stereotyped pruning of the visual and motor CST axon collaterals is differentially regulated and that this specificity arises from the differential expression of plexin receptors in the cortex.axon pruning ͉ corticospinal tract ͉ plexin A functional nervous system depends on the precise wiring of neuronal connections with appropriate targets. During early development, neurons tend to send out axons with excessive branches to multiple target areas. When the neuronal targets become mature, the unnecessary branches are specifically pruned. Stereotyped axon pruning, or pruning of long axon collaterals in a predictable manner, is a major phenomenon in the developing CNS. This type of pruning has been observed in species ranging from Drosophila to mouse and is thought to be essential for the normal development of the CNS (1-5).One classic example of stereotyped pruning in higher vertebrates is in the developing corticospinal tract (CST) (6-10). In developing rodents, CST axons originate from layer V cortical pyramidal neurons in all regions of the neocortex (9, 11). These axons are guided through the internal capsule, cerebral peduncle, and pyramidal tract and then turn dorsally to cross the midline at the pyramidal decussation before they reach the contralateral spinal cord (Fig. 1A). The targeting of primary CST axons to the spinal cord is followed by axon collateral branching to targets in the brainstem and spinal cord (Fig. 1B). This initial projection pattern of CST axons is later modified via stereotyped axon pruning as regions of the neocortex become specialized, and the rostral-caudal location of parent cells within the neocortex determines which axon collaterals are pruned (Fig. 1C). Thus, motor neurons in rostral cortex prune their axons from the superior and inferior colliculi, whereas visual neuron...

Coble JP, Grobe JL, Johnson AK, Sigmund CD. Mechanisms of brain renin angiotensin system-induced drinking and blood pressure: importance of the subfornical organ. Am J Physiol Regul Integr Comp Physiol 308: R238 -R249, 2015. First published December 17, 2014 doi:10.1152/ajpregu.00486.2014.-It is critical for cells to maintain a homeostatic balance of water and electrolytes because disturbances can disrupt cellular function, which can lead to profound effects on the physiology of an organism. Dehydration can be classified as either intra-or extracellular, and different mechanisms have developed to restore homeostasis in response to each. Whereas the renin-angiotensin system (RAS) is important for restoring homeostasis after dehydration, the pathways mediating the responses to intra-and extracellular dehydration may differ. Thirst responses mediated through the angiotensin type 1 receptor (AT 1R) and angiotensin type 2 receptors (AT 2R) respond to extracellular dehydration and intracellular dehydration, respectively. Intracellular signaling factors, such as protein kinase C (PKC), reactive oxygen species (ROS), and the mitogen-activated protein (MAP) kinase pathway, mediate the effects of central angiotensin II (ANG II). Experimental evidence also demonstrates the importance of the subfornical organ (SFO) in mediating some of the fluid intake effects of central ANG II. The purpose of this review is to highlight the importance of the SFO in mediating fluid intake responses to dehydration and ANG II.angiotensin; blood pressure; barin; fluid; renin FLUID BALANCE is crucial for the homeostasis and survival of organisms because they have to properly maintain fluid and electrolyte concentrations for the normal function of all cells. Fluid balance can go awry in pathological states such as in chronic kidney disease and diabetes. Among its many roles in maintaining homeostasis, the renin-angiotensin system (RAS) is an important regulator of fluid balance. The RAS achieves this through the actions of its main effector peptide, angiotensin II (ANG II) through the ANG II type 1 (AT 1 R) and type 2 (AT 2 R) receptors. Activation of AT 1 R by blood-borne ANG II in target tissues causes increases in sodium and water retention, arginine vasopressin (AVP), and aldosterone release, vasoconstriction, increased sympathetic nervous system activity, and increased fluid intake. ANG II is produced by the consecutive enzymatic action of renin on its substrate angiotensinogen (AGT) and by angiotensin-converting enzyme (ACE) on its substrate angiotensin I (ANG I). The renin-AGT cleavage step is generally the rate-limiting step in the production of ANG II, and this is certainly true in the brain where the amount of renin is limiting. The RAS exists in two forms: a circulatory form where ANG II acts as an endocrine factor, and a tissue-specific form, where local production of ANG II acts in an autocrine or paracrine manner to induce AT 1 R signaling on ANG II producing or nearby cells (75). Intracrine, or intracellular forms of the RAS, hav...

Background: The development of the corticospinal tract (CST) in higher vertebrates relies on a series of axon guidance decisions along its long projection pathway. Several guidance molecules are known to be involved at various decision points to regulate the projection of CST axons. However, previous analyses of the CST guidance defects in mutant mice lacking these molecules have suggested that there are other molecules involved in CST axon guidance that are yet to be identified. In this study, we investigate the role of plexin signaling in the guidance of motor CST axons in vivo.

Differential composition of GABAA receptor (GABAAR) subunits underlies the variability of fast inhibitory synaptic transmission; alteration of specific GABAAR subunits in localized brain regions may contribute to abnormal brain states such as absence epilepsy. We combined immunocytochemistry and high-resolution ImmunoGold electron microscopy to study cellular and subcellular localization of GABA AR ␣1, ␣3, and ␤2/␤3 subunits in ventral posterior nucleus (VP) and reticular nucleus (RTN) of control rats and WAG/Rij rats, a genetic model of absence epilepsy. In control rats, ␣1 subunits were prominent at inhibitory synapses in VP and much less prominent in RTN; in contrast, the ␣3 subunit was highly evident at inhibitory synapses in RTN. ␤2/␤3 subunits were evenly distributed at inhibitory synapses in both VP and RTN. ImmunoGold particles representing all subunits were concentrated at postsynaptic densities with no extrasynaptic localization. Calculated mean number of particles for ␣1 subunit per postsynaptic density in nonepileptic VP was 6.1 ؎ 3.7, for ␣3 subunit in RTN it was 6.6 ؎ 3.4, and for ␤2/␤3 subunits in VP and RTN the mean numbers were 3.7 ؎ 1.3 and 3.5 ؎ 1.2, respectively. In WAG/Rij rats, there was a specific loss of ␣3 subunit immunoreactivity at inhibitory synapses in RTN, without reduction in ␣3 subunit mRNA or significant change in immunostaining for other markers of RTN cell identity such as GABA or parvalbumin. ␣3 immunostaining in cortex was unchanged. Subtle, localized changes in GABAAR expression acting at highly specific points in the interconnected thalamocortical network lie at the heart of idiopathic generalized epilepsy.absence epilepsy ͉ inhibition ͉ quantitative electron microscopy T he idiopathic generalized epilepsies are characterized by abrupt losses of consciousness during which the electroencephalogram (EEG) exhibits paroxysmal, high-amplitude spike and wave complexes at Ϸ3Hz and lasting from a few seconds to Ͻ1 min. The loss of consciousness is referred to as an absence seizure or petit mal. Spike and wave activity reflects paroxysmal discharging of neurons in the network of reentrant thalamocortical and corticothalamic connections (1-3). Linkage studies in humans suggest involvement of genes encoding GABA A receptor (GABA A R) subunits of the ␣, ␤, ␥, and ␦ families in absence epilepsy (4-7), and experimental studies in animals point to molecular genetic abnormalities in GABA A R signaling (8, 9). The forms by which genetic anomalies in GABA A R manifest themselves are multiple and varied. They can involve receptor synthesis and trafficking within neurons, changes in ratios of alternatively spliced mRNAs, and translocations of receptor subunits to membrane sites not normally occupied (10-14).The GABAergic neurons of the thalamic reticular nucleus (RTN) play a key role in synchronizing activity in the thalamocortical network during states of consciousness (15, 16), and GABA A R antagonists applied to its cells can transform 7-to 14-Hz sleep spindle oscillations generated in the ...

Increased activity of the renin-angiotensin system within the brain elevates fluid intake, blood pressure, and resting metabolic rate. Renin and angiotensinogen are coexpressed within the same cells of the subfornical organ, and the production and action of ANG II through the ANG II type 1 receptor in the subfornical organ (SFO) are necessary for fluid intake due to increased activity of the brain renin-angiotensin system. We generated an inducible model of ANG II production by breeding transgenic mice expressing human renin in neurons controlled by the synapsin promoter with transgenic mice containing a Cre-recombinase-inducible human angiotensinogen construct. Adenoviral delivery of Cre-recombinase causes SFO-selective induction of human angiotensinogen expression. Selective production of ANG II in the SFO results in increased water intake but did not change blood pressure or resting metabolic rate. The increase in water intake was ANG II type 1 receptor-dependent. When given a choice between water and 0.15 M NaCl, these mice increased total fluid and sodium, but not water, because of an increased preference for NaCl. When provided a choice between water and 0.3 M NaCl, the mice exhibited increased fluid, water, and sodium intake, but no change in preference for NaCl. The increase in fluid intake was blocked by an inhibitor of PKC, but not ERK, and was correlated with increased phosphorylated cyclic AMP response element binding protein in the subfornical organ. Thus, increased production and action of ANG II specifically in the subfornical organ are sufficient on their own to mediate an increase in drinking through PKC.

Angiotensin-II production in the subfornical organ acting through angiotensin-II-type-1 receptors is necessary for polydipsia resulting from elevated renin-angiotensin system activity. Protein kinase C and mitogen-activated protein kinase pathways have been shown to mediate effects of angiotensin-II in the brain. We investigated mechanisms that mediate brain angiotensin-II-induced polydipsia. We used double transgenic sRA mice, consisting of human renin controlled by the neuron-specific synapsin promoter crossed with human angiotensinogen controlled by its endogenous promoter, which results in brain-specific overexpression of angiotensin-II, particularly in the subfornical organ. We also employed the deoxycorticosterone acetate-salt model of hypertension which exhibits polydipsia. Inhibition of protein kinase C, but not extracellular signal-regulated kinases, protein kinase A, or vasopressin V1A and V2 erceptors, corrected the elevated water intake of sRA mice. Using an isoform selective inhibitor and an adenovirus expressing dominant negative protein kinase C-α revealed that protein kinase C-α in the subfornical organ was necessary to mediate elevated fluid and sodium intake in sRA mice. Inhibition of protein kinase C activity also attenuated polydipsia in the deoxycorticosterone acetate-salt model. We provide evidence that inducing protein kinase C activity centrally is sufficient to induce water intake in water-replete wildtype mice, and that cell surface localization of PKC-α can be induced in cultured cells from the subfornical organ. These experimental findings demonstrate a role for central protein kinase C activity in fluid balance and further mechanistically demonstrate the importance of protein kinase C-α signaling in the subfornical organ in fluid intake stimulated by angiotensin-II in the brain.

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Transgenic mice expressing human renin via the neuron‐specific synapsin promoter (sR) were bred with ubiquitous CAG promoter, conditionally inducible human angiotensinogen (AGT) mice (ARed). Recombination of the ARed transgene by Cre‐recombinase results in loss of a dsRed marker and gain of AGT expression. Adenovirus encoding Cre (AdCre) was injected into the lateral cerebral ventricle (ICV) of sRARed and littermate control mice. At baseline, total fluid and sodium intakes of sRARed mice (0.30±0.02 mL/day; 0.97±0.15 mEq/day, n=12) was similar to littermates (0.24±0.03 mL/day; 0.63±0.03 mEq/day, n=16) in a two‐bottle choice paradigm (water vs 0.15 M NaCl). Three weeks after AdCre injection, sRARed mice increased both total fluid and sodium intakes in comparison to baseline (0.46±0.05 mL/day, P=0.01; 1.79±0.20 mEq/day, P<0.01, n=12) and littermate controls (0.27±0.04 mL/day, P<0.01; 1.03±0.11 mEq/day, P<0.01, n=16). Mice expressing a reporter construct sensitive to Cre‐recombination (ROSA‐tdTomato) were injected using the same methodology as the sRARed mice. ICV delivery of AdCre into ROSA‐tdTomato mice resulted in reporter gene expression throughout the ependyma but also within the subfornical organ and median preoptic nucleus. We developed a model of conditionally inducible expression of AGT, which can be used to temporally and spatially control the production of angiotensin peptides in the brain.