To migrate along the vasculature, glioma cells must first contact individual vessels. Studies of transplanted C6 rat glioma cells into rat forebrain suggest that migrating glioma cells come into contact with the vasculature in less than 24 h (Farin et al. 2006) . One mechanism by which glioma cells chemotactically migrate towards the vasculature is by responding to bradykinin , a kinin released by vascular endothelial cells (Montana and Sontheimer 2011) . Bradykinin binds to the B2 receptor constitutively expressed by human glioma cells (Montana and Sontheimer 2011). When human glioma cells were placed on murine brain slices, in 2 h bradykinin induced nearly 85 % of glioma cells to contact blood vessels; this interaction was reduced to 30 % when the B2 receptor was selectively knocked down in human glioma cells (Montana and Sontheimer 2011). Bradykinin application also increased the total number of migrating glioma cells, and increased the speed and distance travelled by individual glioma cells (Montana and Sontheimer 2011), further emphasizing the importance of this molecular interaction.

Once contacting the vasculature, glioma cells migrate in a saltatory manner at a speed of about 6–25 µm/h (Farin et al. 2006; Turner and Sontheimer 2013; Watkins and Sontheimer 2011) . Contact is maintained with the vasculature through activation of α-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptors on the leading edges of migrating glioma cells, which promote binding to extracellular matrix components, including type I and type IV collagen found in perivascular areas (Piao et al. 2009) . Increased AMPA receptor expression correlated with enhanced expression of β1-integrins on the plasma membrane and increased numbers of focal adhesion complexes, all of which are involved in attachment of glioma cells to extracellular substrates, promoting glioma cell invasion (Piao et al. 2009).

The vasculature of the brain is unlike the rest of systemic circulation for at least two principal reasons: (1) tight junctions between vascular endothelial cells form a blood-brain barrier , and (2) the “neurovascular unit,” composed of vascular endothelial cells, astrocytes, and neurons, couples neuronal activity to vascular perfusion. Maintenance of the blood-brain barrier is important, as it prevents ions, molecules, and cells from non-selectively infiltrating the brain, where the extracellular milieu is tightly regulated for optimal neuronal functioning. One mechanism by which neuronal activity leads to enhanced vascular perfusion is through the spillover of glutamate from synapses onto adjacent astrocytes. This glutamate can activate astrocytic metabotropic glutamate receptors (mGluR), which through a cascade involving G-protein coupled signaling lead to downstream increases in intracellular [Ca²⁺], which in turn leads to release of arachidonic acid metabolites and K⁺, subsequently modulating of vascular tone (Drake and Iadecola 2007; Attwell et al. 2010) . An increase in cerebral blood flow in response to increased neuronal activity is termed “functional hyperemia” and serves to enhance delivery of O₂ and glucose to and remove waste metabolites from hyper-metabolic brain regions.

As glioma cells migrate along the vasculature, the native cytoarchitecture is disrupted, opening the blood-brain barrier. Implantation of C6 rat glioma cells into the cerebrum of rats demonstrated that the processes and cell bodies of migrating glioma cells intercalate between vascular endothelial cells and astrocytic endfeet (Farin et al. 2006) . Tumor cells actively “lift up” astrocytic processes from the vasculature as revealed by transmission electron microscopy (Zagzag et al. 2000) . After movement of the astrocytic processes, glioma cells can directly attach to the perivascular basement membrane and cause the perturbed astrocytes to become “reactive” (Nagano et al. 1993) , which in turn can lead to downstream ramifications including loss of functional hyperemia and impairment of neuronal function . Incidentally, the association of glioma cells with the vasculature also seems to promote proliferation; more than 60 % of glioma cells that divided were less than 20 µm from a vascular branch point (Farin et al. 2006) .

Migrating human glioma cells shrink in volume by 33 % to move through narrow extracellular spaces irrespective of the size of the cell or spatial constraint (Watkins and Sontheimer 2011) . This dramatic decrease in cellular volume is accomplished through the efflux of Cl⁻ ions into the extracellular space, leading to the obligated release of cytoplasmic water (Watkins and Sontheimer 2011) . Glioma cells accumulate large concentrations of Cl⁻, up to 100 mM, in the cytoplasmic space (Habela et al. 2009), likely due to the activity of NKCC1, a Cl⁻ cotransporter highly expressed by human glioma cells (Haas and Sontheimer 2010) . The high intracellular [Cl⁻]_i provides a large driving force for Cl⁻ efflux, thereby moving water across the plasma membrane.

ClC-3 , a voltage-gated Cl⁻ channel, has been identified as a key mediator of Cl⁻ efflux by human glioma cells. ClC-3 is highly expressed on the plasma membrane (Olsen et al. 2003) and is activated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) (Huang et al. 2001; Cuddapah and Sontheimer 2010) , a Ca²⁺-sensitive kinase. CaMKII-dependent activation of ClC-3 is critical for glioma cell migration , as its inhibition either though pharmacological or genetic manipulation prevents glioma cell movement through spatial barriers (Cuddapah and Sontheimer 2010; Cuddapah et al. 2013a) . ClC-3 mediated Cl⁻ flux is coupled to movement of K⁺ into the extracellular space through 2 Ca²⁺-activated K⁺ channels: K_Ca3.1 and K_Ca1.1 (Cuddapah et al. 2013a; Weaver et al. 2006) . Coupling of Cl⁻ to K⁺ movement may allow migrating cells to maintain electroneutrality.

Intracellular [Ca²⁺] in gliomas increases secondary to a variety of ligands and channels, including glutamate , bradykinin , epidermal growth factor (EGF), lysophosphatidic acid (LPA), or transient receptor potential (TRP) channels, all of which enhance migration and invasion (Lyons et al. 2007; Ishiuchi et al. 2002; Montana and Sontheimer 2011; Bomben et al. 2011; Cuddapah et al. 2013a, 2013b; Manning et al. 2000) . Once Ca²⁺ increases, ion channels as well as cytoskeletal elements are regulated to promote migration. Specifically, three integral components of migration, including protrusion, attachment, and retraction, are activated. Protrusion involves the lamellipodium, rich with polymerizing actin filaments, which pushes the plasma membrane forward (Fig. 14.3a, 14.3b). Through a well-established treadmilling model, actin monomers away from the lamellipodium are depolymerized and actin monomers in the lamellipoium are polymerized, pushing the leading edge forward. This highly coordinated process requires Ca²⁺ signaling; Ca²⁺ signaling acts on Rho GTPases leading to downstream regulation of actin depolymerizing factor/cofilin family of proteins (Zheng and Poo 2007) . Additionally, focal adhesions, which bind migrating cells to the substratum and provide contact points to build traction, are regulated by CaMKII (Easley et al. 2008) . Thus Ca²⁺ dynamics are involved in the protrusion of the lamellipodium and attachment to the substratum (Fig. 14.3b). Importantly, increases in intracellular [Ca²⁺] also activate ion channels, which flux osmotically-active ions to move cytoplasmic water across the plasma membrane (Fig. 14.3c), allowing the motile cell to change shape and volume. For example, bradykinin-mediated Ca²⁺ elevations can lead to K_Ca 3.1 and CaMKII-dependent ClC-3 channel activation in human glioma cells (Cuddapah et al. 2013a) . This ion channel activation leads to shape and volume changes, and if inhibited, blocks migration (Watkins and Sontheimer 2011) . At the lagging edge myosin II contracts pushing the cell forward (Yang and Huang 2005; Conrad et al. 1993) (Fig. 14.3d). Given that a variety of ligands enhance glioma cell migration through an increase in Ca²⁺, this may be a convergent pathway common to multiple upstream signaling mechanisms.

Similar to glioma cell migration , the division of glioma cells requires coordinated shape and volume changes. As glioma cells enter mitosis and prepare to divide into two daughter cells, cellular shape changes from bipolar to round (Cuddapah et al. 2011; Habela and Sontheimer 2007; Habela et al. 2008). The importance of a round morphology seems manifold, including allowing the cellular membrane and organelles to split symmetrically, and stabilizing the mitotic spindle for equal distribution of genomic content (Rosenblatt 2008) . This cellular rounding is associated with a simultaneous decrease in cytoplasmic volume by approximately 40 % termed pre-mitotic condensation (Habela and Sontheimer 2007; Habela et al. 2009). Importantly, CaMKII activation of ClC-3 has been identified as a mechanism that promotes pre-mitotic condensation in human glioma cells. Dividing glioma cells have a large Cl⁻ conductance mediated by CaMKII activation of ClC-3 (Habela et al. 2008; Cuddapah et al. 2011). As in the context of migration, the large Cl⁻ conductance in dividing human glioma cells is due to efflux of osmotically-active Cl⁻ ions, which is sufficient to condense cytoplasmic volume by 33 % (Habela et al. 2009). Inhibition of ClC-3 or CaMKII significantly increases the time required for cytoplasmic condensation, thereby decreasing glioma cell proliferation by 35 % (Cuddapah et al. 2011).

This hydrodynamic process of pre-mitotic condensation may be coupled to other Ca²⁺-dependent processes to ensure robust proliferation in human glioma cells. During the first phase of mitosis, prophase, cyclin B binds to Cdk1 to promote cell division (Fig. 14.4a). The replicated chromosomes then begin to condense inside an intact nuclear envelope. Additionally, the replicated centrosomes begin to migrate to opposite poles of the cell. Between prophase and metaphase is prometaphase, which includes several steps preceding formation of the metaphase plate (Fig. 14.4b). The nuclear envelope breaks down, and the replicated chromosomes attach to microtubules through their kinetochores. The microtubules, in turn, are attached to centrosomes, forming the mitotic spindle. Ca²⁺ and calmodulin levels increase around the centrosome and microtubules (Welsh et al. 1979; Wolniak et al. 1983) to stabilize tubulin polymerization and depolymerization. As the cell transitions into metaphase, both centrosomes, already located at opposite poles of the cell, pull on the chromosomes (Fig. 14.4c). This tension generated by the opposing centrosomes pulls the chromosomes to the center of the cell forming the metaphase plate. The symmetric microtubule tension also drastically changes the cytoarchitecture leading to cellular shape changes. The metaphase cell begins to become round characterizing the process known as mitotic cell rounding. Towards the end of metaphase, intracellular [Ca²⁺] spikes for about 20 s (Ratan et al. 1988; Poenie et al. 1986) , potentially activating CaMKII and ClC-3 on the plasma membrane. As detailed above, because there is a large driving force for Cl⁻ efflux (Habela et al. 2009), opening of ClC-3 channels leads to Cl⁻ loss, subsequently driving the osmotic loss of cytoplasmic water. This cytoplasmic condensation leads to a shrinking of cellular volume, bridging kinases with their substrate proteins. After the intracellular [Ca²⁺] spike returns to baseline, daughter chromatids are separate to opposite ends of the cell towards the centrosomes, characterizing anaphase (Fig. 14.4d). As during prometaphase, Ca²⁺ and calmodulin levels remain elevated around the mitotic spindle, facilitating tubulin polymerization and depolymerization. This model suggests that Ca²⁺ is a master regulator and drives several necessary events between the metaphase-anaphase transition, and if disrupted, hinders mitotic progression. Ion channels play a critical role in mitosis by facilitating the dynamic shape and volume changes characteristic of dividing cells.

One of the key functions of astrocytes is to take up glutamate from the extracellular space to dampen neuronal excitability. This glutamate uptake is primarily achieved through plasma membrane Na⁺-dependent glutamate transporters including GLT-1 and GLAST (in rodents; in human, EAAT2 and EAAT1, respectively). In contrast, gliomas, the malignant counterpart of astrocytes, fail to take up glutamate from the extracellular space (Ye and Sontheimer 1999) . Na⁺-dependent glutamate uptake is up to 100-times less in human glioma cells, secondary to decreased GLT-1 expression and mislocalization of GLAST to the nucleus (Ye et al. 1999) . Instead, glutamate is persistently released through the system xc- cystine-glutamate exchanger. Cystine enters the cell and is converted to glutathione, an antioxidant critical for the absorption of free radicals formed by gliomas in hypoxic environments (Ogunrinu and Sontheimer 2010) . Glutamate released in the extracellular space (1) acts in an autocrine loop on gliomas to increase tumor aggressiveness, and (2) induces hyperexcitability in peritumoral neurons and eventual excitotoxic cell death.

Human glioma cells lack the GluA2 subunit in endogenously expressed AMPA receptors, rendering the channels Ca²⁺-permeable (Ishiuchi et al. 2002, 2007; Lyons et al. 2007) . Therefore, when glutamate is released from the system xc-, it then acts in an autocrine/paracrine manner to increase Ca²⁺ signaling in glioma cells, which correlates with increased migration (Ishiuchi et al. 2002; Lyons et al. 2007) . Inhibition of glutamate release from the system xc- or inhibition of Ca²⁺-permeable AMPA receptors decreased glioma cell migration and reduced overall tumor growth (Lyons et al. 2007). Additionally, conversion of AMPA channels from Ca²⁺-permeable to Ca²⁺-impermeable by overexpression of the GluA2 subunit significantly decreased tumor volume by inhibiting glioma proliferation and invasiveness and increasing glioma cell apoptosis (Ishiuchi et al. 2002). Mechanistically, Ca²⁺ permeation through AMPA receptors may promote proliferation and migration through the activation of Ca²⁺-sensitive ion channels that facilitate hydrodynamic shape and volume changes (Cuddapah et al. 2011, 2013a) . Alternatively, Ca²⁺ influx may activate Akt, thereby enhancing signaling cascades that lead to proliferation and migration (Ishiuchi et al. 2007).

Beyond autocrine/paracrine signaling, glioma-released glutamate acts on neurons to induce hyperexcitability and eventual neuronal death. Human gliomas implanted into mouse cerebrums induced epileptic activity within 14–18 days, as measurable by electroencephalogram (Buckingham et al. 2011) . This hyperexcitability was due to glutamate release from the system xc- acting at peritumoral neurons (Buckingham et al. 2011). Importantly, from a clinical perspective, pharmacological inhibition of the system xc- reduced neuronal hyperexcitability and seizure activity (Buckingham et al. 2011; Campbell et al. 2012) .

As extracellular glutamate concentrations increase in the peritumoral environment, neuronal hyperexcitability snowballs into excitotoxicity . Abnormal glutamate activation of ionotropic glutamate receptors leads to excessive Ca²⁺ influx, leading to the activation of phospholipases and proteases associated with excitotoxicity. Incubation of neurons in media exposed to human glioma cells is sufficient to cause sustained Ca²⁺ increases in neurons, characteristic of excitotoxicity (Ye and Sontheimer 1999) . This leads to neuronal death and can be reversed by blocking Ca²⁺-permeable N-methyl-D-aspartate (glutamate) receptors with MK-801 or by inhibiting glutamate release from glioma cells (Ye and Sontheimer 1999).

Cerebral vascular abnormalities typify many neurodegenerative diseases , and malignant gliomas certainly meet this criterion. Neurodegeneration, including neuronal loss and deficits in memory and learning, can directly result from poor microvascular circulation secondary to ablation of pericytes, an important component of the neurovascular unit (Bell et al. 2010) . Additionally, mutations in superoxide dismutase 1 (SOD1), which can lead to ALS, cause disruptions in the blood-spinal cord barrier by inducing downregulation of several endothelial tight junction proteins including ZO-1 and occludin (Zhong et al. 2008) . Interestingly, the breakdown of the blood-spinal cord barrier precedes motor neuron loss in the ventral horn that is typical of the disease (Zhong et al. 2008) , suggesting the vascular breakdown plays a causative role in neurodegeneration. Alzheimer’s disease is also associated with disrupted vascular tone as amyloid beta deposits lead to disorganization of vascular smooth muscle cells (Christie et al, 2001). When gliomas are implanted in to mice overexpressing amyloid beta, tumors are less vascularized, demonstrating the inherent antiangiogenic properties of amyloid beta (Paris et al. 2010) . Similar to other neurodegenerative conditions, gliomas are characterized by a malfunctioning blood-brain barrier and poor vascular perfusion. Glioma cells migrate along the vasculature, disrupting native cytoarchitecture, and induce angiogenesis, leading to the growth of leaky and hypo-functional vessels. Dysfunctional vessels may lead to a loss of coupling between neuronal firing and vascular perfusion and yield focal symptoms associated with gliomas.