Every member of the TRPC subfamily is expressed in the brain (Figure 23.1), some (like TRPC1) ubiquitously, whereas others (e.g., TRPC4) in a highly restricted pattern [1]. Further details are out of the scope of this chapter (interested readers are referred to [2–4]). In general, TRPCs (a) regulate neural stem cell proliferation; (b) promote neuronal cell survival in response to neurotrophins such as BDNF; (c) are involved in chemoattraction of neurites, growth cone guidance and regulation of neurite outgrowth spine formation; and (d) are increasingly accepted as major players in synaptic plasticity and synaptogenesis (Figure 23.2; see 4 for a review]. These functions are especially prominent in the developing brain.

Vanilloid TRP Channels

Of the vanilloid TRP subfamily, the presence of TRPV1 to V4 has been reported in the brain (Figure 23.1). The pharmacology of these channels have been detailed elsewhere [2].

The Ankyrin TRPA1 Channel

In the rat brain, TRPA1 is expressed in neurons, astrocytes, and cells lining the choroid plexus and the ventricles. In the hippocampus, TRPA1 has been implicated in neuronal cell death. The synthetic cannabinoid WIN 55,212-2 protects hippocampal neurons against ischemic damage. This protective action was suspended by the CB1 receptor antagonist AM251, but not the CB2 receptor antagonist AM630. The TRPA1 blocker HC-030031 enhanced the neuroprotective efficacy of WIN 55,212-2. In contrast, the TRPA1 agonist icilin or allyl isothiocyanate led to a stronger neurodegeneration. These data suggest that WIN 55,212-2 has a biphasic action: at low concentrations it protects neurons by activating CB1 receptors, and at high concentrations it additionally activates TRPA1 that interferes with the CB1 receptor-mediated neuroprotection [119].

Somewhat confusingly, TRPA1 plays a protective role in astrocytes. In these glial cells, H₂S₂ generates polysulfides that, in turn, induce Ca^2 + influx. This effect is prevented by the TRPA1 antagonist HC-030031, as well as by Trpa1 gene silencing via siRNAs [120]. In, addition, astrocytic TRPA1 contributes to basal Ca^2 + levels that are required for constitutive release of d-serine, a signaling molecule used to communicate with neurons [121].

TRPA1 is expressed in both the choroid plexus and ventricular lining epithelium [122], as well as in magnocellular neurosecretory cells of the supraoptic nucleus that produce vasopressin [123]. Given its role as a chemosensor, one might speculate that TRPA1 may respond to chemical stimuli to regulate CSF synthesis and vasopressin release.

TRPC Channel and Epilepsy

The muscarinic agonist, pilocarpine, evokes seizures in experimental animals; this is a widely used model of temporal lobe epilepsy. Severe pilocarpine-induced seizures may cause neuronal cell death in the lateral septum; this effect is ameliorated in the Trpc1/c4 double knockout mouse. These results imply an essential role for TRPC1 and TRPC4 (likely as a heteromultimer) in forming the intrinsic membrane conductance that mediates the plateau potential in lateral septal neurons [124,125].

The Trpc5 knockout mouse also exhibits both significantly reduced seizure activity and attenuated seizure-induced neuronal cell death in the hippocampus. Yet, epileptiform bursting activity induced by mGluR agonists (which is normal in the Trpc1/c4 double knockout animals) is unaltered in Trpc5 null mice. By contrast, long-term potentiation is (which is normal in the Trpc1/c4 double knockout animals) is greatly reduced in TRPC5-deficient mice. The distinct phenotype of these knockout animals suggests that TRPC5 and TRPC1/C4 contribute to seizure activity by distinct cellular mechanisms [124]. In support of this model, muscarinic activation by carbachol in hippocampal neurons was shown to initiate a current through TRPC5 by promoting the membrane insertion of the channel. As expected, the muscarinic antagonist, atropine, prevented the increase in TRPC5 surface expression. TRPC5-like currents were also inhibited by the nonselective TRPC antagonists, 2-APB and SKF-96365, as well as the PI3K inhibitor, wortmannin, which blocks TRPC5 translocation from the cytosol to the plasma membrane. In conclusion, the rapid translocation of TRPC5 contributes to the generation of the cholinergic-induced plateau potentials through a Ca^2 +/calmodulin and PI3K-dependent pathway, providing new insights into the pathology of epilepsy [126].

In the motor cortex, TRPC3 expression is strong during embryogenesis but is weak to absent in the mature brain. In dysplastic cortex, TRPC3 expression, however, returns to the high levels seen in developing neurons. In pyramidal neurons, combinations of low Ca^2 + and Mg^2 + increases the amplitude of depolarization, which, in the dysplastic cortex, is sufficient to provoke epileptiform activity. Importantly, this activity was suppressed by the TRPC3 inhibitor, Pyrazole-3 (Pyr3) [127]. Following status epilepticus, TRPC3 expression is elevated (whereas TRPC6 is reduced) in pyramidal neurons and dentate granule cells. This increase in TRPC3 is blocked by Pyr3. Furthermore, hyperforin (a TRPC6 activator) prevents down-regulation of TRPC6 induced by epileptic seizures. Apparently, both increased TRPC3 and decreased TRPC6 are relevant because both Pyr3 and hyperforin (alone or in combination) protected neurons against damage by severe epileptic seizures [128]. Interestingly, status epilepticus induced TRPC3 expression in endothelial cells that did not contain this protein in control animals. The neo-expression of TRPC3 in endothelial cells was accompanied by a loss of SMI-71, a blood-brain barrier marker, and correlated to the development of vasogenic edema and resultant neuronal damage. The vasogenic edema response was attenuated by Pyr3. It was speculated that TRPC3 neo-expression in endothelial cells may contribute to the neuronal damage during and after status epilepticus by disrupting the blood-brain barrier [129].

In summary, TRPC channels could represent shared downstream target for a number signaling pathways that may contribute to seizure and excitotoxicity, such as NMDA receptor-mediated Ca^2 + influx, or mGluR activation [130]. Of TRPC channels, TRPC3 is particularly interesting given its preponderance in dysplastic (and low expression in normal) motor cortex and postulated role in disrupting the blood-brain barrier during status epilepticus.

TRPV1 as a Potential Target in Epilepsy and Febrile Seizures

TRPV1 is an interesting, though highly controversial (the ongoing debate whether or not TRPV1 is expressed at all in the brain is discussed elsewhere), antiepileptogenic target. In rats, TRPV1 activation was reported to modulate activity-dependent synaptic efficacy in the hippocampus by facilitating LTP and suppressing LTD. In brain slices obtained from Trpv1 (−/−) mice, LTD was absent, and capsaicin was inefficient [131,132]. It was hypothesized that TRPV1 selectively inhibits excitatory synapses at hippocampal interneurons. 4-aminopyridine triggers epilepsy-like symptoms in the rat. In this model, the first-generation TRPV1 antagonist, capsazepine, suppresses epileptiform activity. By contrast, capsaicin potentiates the seizures [133]. To further strengthen the link between TRPV1 and epilepsy, it was pointed out that NGF (a known driver of TRPV1 expression) triggers epileptogenesis. Furthermore, the levels of the endocannabinoid anandamide (also an endogenous TRPV1 agonist) are increased in epilepsy. It was speculated that TRPV1 activation (possibly by high anandamide concentrations) may trigger apoptotic neuronal death (at least in the rat brain) that leads to chronic epilepsy [134].

The cannabinoid link to epilepsy is a hot topic. Those who favor legalization point out that medical marijuana (or its main active ingredient, tetrahydrocannabinol) can control seizures in experimental animals not responsive to other treatments. Also, there are anecdotal reports that medical marijuana can stop intractable seizures in children with Duvet syndrome, also known as myoclonic epilepsy of infancy. Boosting endogenous anandamide in the brain by blocking FAAH (the enzyme that hydrolyzes anandamide) has anticonvulsant effects, which are mediated by CB1 receptors. The trick is to keep anandamide in the beneficial dose range because high anandamide concentrations can cause paradoxical seizures by activating TRPV1 [135,136]. One strategy to achieve this goal is by dual FAAH and TRPV1 blockade with N-arachidonoyl-serotonin (AA-5-HT). Indeed, seizures induced by pentylenetetrazole in mice were prevented by AA-5-HT [137].

Importantly, there is experimental evidence linking TRPV1 to epilepsy in humans. Both tuberous sclerosis complex and focal cortical dysplasia type IIb cause intractable epilepsy. In brain specimens removed during surgery from patients with tuberous sclerosis or focal cortical dysplasia IIb, TRPV1 was detected in the abnormal cell types, such as dysmorphic neurons, balloon cells, and giant cells. Interestingly, TRPV1 appeared to have both cytoplasmic and nuclear distribution, suggesting a potential nuclear role of TRPV1 [138]. Elevated TRPV1 was also detected in the brains of patients with mesial temporal lobe epilepsy compared to controls (brain surgery unrelated to epilepsy). TRPV1 was mainly present in the cell bodies and dendrites of glutaminergic and GABAergic neurons and GFAP-positive astrocytes [139]. At present, it is unclear if this abnormal TRPV1 expression is a cause or consequence of the disease.

As a heat-activated channel, TRPV1 seems to be an interesting candidate to explore for mediating febrile seizures, the most common seizure type in children under the age of five. In mice, pentylene tetrazol induces clonic seizures that are made worse in febrile animals. In this model, Trpv1 gene deficiency decreased the intensity of experimental febrile seizures [140]. In rats treated with subconvulsive doses of pentylene tetrazol, the TRPV1 receptor agonist OLDA (injected intracerebroventricularly 30 min prior to pentylene tetrazol administration) accelerated the incidence of seizures, whereas the TRPV1 antagonist AMG-9810 ameliorated the seizure activity [141].

TRPM2 and Juvenile Myoclonic Epilepsy

Mutations in the gene that encodes the EF-hand motif-containing protein EFHC1 have been linked to the pathobiology of juvenile myoclonic epilepsy (JME). In hippocampal neurons, TRPM2 and EFHC1 are coexpressed with a physical interaction between the N- and C-terminal cytoplasmic regions of TRPM2 with the EFHC1 protein. In recombinant TRPM2 expression systems, coexpression of EFHC1 was shown to potentiate the H₂O₂-induced currents with a resultant increase in cell death. Based on these findings it was speculated that TRPM2 mediates the disruptive effects of JME mutations of EFHC1 [142].

In animal models, neurotoxins (e.g., rotenone, a pesticide) that kill dopaminergic neurons in a Ca^2 + overload-dependent manner mimic some aspects of the human disease. The rise in [Ca^2 +]_i evoked by rotenone was attenuated by the TRPM2 blocker, N-(p-amylcinnamoyl) anthranilic acid [149]. In SH-SY5Y cells, salsolinol (a toxic condensation product of dopamine and acetaldehyde) and 1-methyl-4-phenylpyridinium (MPP, a neurotoxin that causes parkinsonism in primates) induce apoptosis, which is reversed by TRPC1 activation. Exposure of SH-SY5Y cells to these toxins blocks thapsigargin-mediated Ca^2 + influx and decreases TRPC1 in the plasma membrane by translocation to the cytoplasm. It was speculated that neurotoxins may alter Ca^2 + homeostasis (in a way that involves TRPC1 possibly via PLC) and induce mitochondrial-mediated caspase-dependent cytotoxicity, an important characteristic of PD [150].

Unexpectedly, capsaicin microinjected into the substantia nigra provides partial protection against neuronal cell death induced by MPP by suppressing the production of microglia-derived ROS. These capsaicin effects are reversed by capsazepine. Bases on these findings, it was speculated that TRPV1 agonists may have a therapeutic value in PD [151]. (How one can safely deliver a TRPV1 agonist into the CNS is a completely different problem, not addressed in this article!)

GABAergic interneurons that express TRPC3 control the activity of striatal projection neurons. Parkinsonian movement disorders are often associated with abnormalities in the firing intensity and/or pattern of these cells. TRPC3 channels expressed by GABAergic interneurons are tonically active and mediate an inward, Na⁺-dependent current, leading to a substantial depolarization. Conversely, inhibition of TRPC3 channels induces hyperpolarization and thereby decreases the firing frequency [152].

The mainstay of pharmacotherapy in PD is dopamine replacement. Unfortunately, the long-term use of levodopa (l-DOPA) as a side effect causes abnormal involuntary movements, called L-DOPA-induced dyskinesia. In a hemiparkinsonian model of PD (mice with 6-hydroxydopamine-induced striatal lesions), chronic L-DOPA treatment leads to the development of intense axial, forelimb, and orolingual dyskinetic symptoms, resembling the human adverse effect. Treatment with oleoylethanolamide (OEA) ameliorated these symptoms without altering the therapeutic motor effects of L-DOPA. The antidyskinetic action of OEA was most likely mediated by TRPV1 because it was absent in mice desensitized to capsaicin [153].

Excessive glutamate can cause neuronal dysfunction and degeneration. Glutamate excitotoxicity has been implicated in acute brain insults (such as ischemic and traumatic brain injury that are discussed later) and also in chronic neurodegenerative disorders including PD. 2-APB (a “universal” TRP channel blocker) reduces glutamate-induced cell death in hippocampal organotypic slice cultures presumably by inhibiting TRPC1 channels. In support of this hypothesis, knockdown by iRNA of Trpc1 in slice cultures prevents glutamate-induced cell death [154]. Apparently, in these cells TRPC1 is an important downstream target for mGluRs [155].

Lou Gehrig’s Disease and Amyotrophic Lateral Sclerosis-Parkinson Dementia Complex

Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s disease) is a progressive neurodegenerative disorder that predominantly affects the spinal motor neurons, leading to muscle weakness and eventual paralysis. The etiology of ALS is unknown but a subset of cases (~ 10%) appears to be inherited. In 1993, mutations in the gene that encodes the SOD1 protein (superoxide dismutase-1, alternatively referred to as superoxide dismutase [Cu^2 +-Zn^2 +]), have been linked to familial ALS. Sod1 (G93A) mutant transgenic mice represent an animal model of ALS. In these mice, increased expression of TRPV4 was noted in the cerebral cortex, hippocampal formation, thalamus, cerebellum, and spinal cord. Both in the cerebral cortex and the hippocampus, TRPV4 was especially increased in the pyramidal cells. It was speculated that TRPV4 may be involved in the pathogenesis of ALS, but the functional implications of increased TRPV4 remain unclear [156].

Amyotrophic lateral sclerosis-Parkinson dementia complex (ALS-PDC) is a mysterious neurodegenerative disorder that combines symptoms of ALS, Parkinsonism, and Alzheimer disease. ALS-PDC is prevalent in the Guam population but is a rarity elsewhere [157]. The etiology of ALS-PDC remains unknown, but the extremely limited geographic distribution of the disease strongly suggests an environmental factor specific to Guam. It was speculated that β-N-methyl-amino alanine (BMAA) may be the toxic agent that damages the neurons to cause ALS-PDC. BMAA is a mixed mGluR agonist present in the cycad plant, a traditional food source in Guam, which causes reversible membrane depolarization and initiates a rise in [Ca^2 +]_i in dopaminergic neurons. The inward current was mainly mediated by mGluR1 via TRPC channels. Indeed, the TRP channel blockers, SKF 96365 and ruthenium red (RR), reduced the BMAA-induced current. Prolonged exposure to BMAA leads to Ca^2 + overload, cell shrinkage, massive cytochrome-c release into the cytosol, and ROS production; ultimately, these changes kill the treated neurons [158].

There is increasing evidence that L-BMAA per se is not sufficient to cause ALS-PDC; another environmental factor is needed. Recent studies have focused on drinking water, which has a unique mineral composition in these Pacific islands; it is extremely low in Ca^2 + and Mg^2 +, but rich in metals like Mn^2 +, Al^3 +, and Fe^3 +. Indeed, rats fed diets that mimic the mineral composition of drinking water in Guam showed significant loss of nigral dopaminergic neurons. A TRPM7 missense mutation (T1482I) was also identified in a subset of ALS-PDC patients [159]. Recent clinical studies, however, have questioned the relevance of the TRPM7 T1482I variant in ALS-PDC. Parametric linkage analyzes of the TRPM7 locus in a large extended family with ALS-PDC patients did not reveal any evidence supporting the linkage to the TRPM7 locus. Resequencing of the entire coding region of TRPM7 did not reveal any pathogenic mutations in an affected individual in this family. The allele frequencies of the T1482I in affected individuals in this family or in those from other families are not significantly different from those in regional controls in Japan [160].

Alzheimer Disease

Alzheimer disease (AD) is the most common form of dementia. Although some cases appear to be familial (and have an early onset), the most important risk factor is advanced age. Indeed, after age 85 the risk for developing AD approaches 50%. Hyperphosphorylated tau aggregated into neurofibrillary tangles is a hallmark lesion of AD. In animal models, cold water exposure causes reversible tau hyperphosphorylation, associated with cognitive deficits. In rats, intragastric capsaicin (10 mg/kg) was reported to mitigate the cognitive decline induced by cold water exposure. Furthermore, capsaicin attenuated the cold water stress-induced spatial memory impairment and prevented tau hyperphosphorylation [161]. These findings are puzzling because capsaicin absorbed from the GI tract undergoes extensive hepatic metabolism and is unlikely to reach pharmacologically meaningful concentrations in the CNS.

In the rat, TRPV4 is highly expressed in hippocampal astrocytes where it is thought to play a major role in oxidative stress-induced cell damage. In aged monkey, circulating amyloid- β40 peptide crosses the blood-brain barrier and is deposited in cerebrovascular endothelial cells, causing amyloid angiopathy. In rat hippocampal slices, synthetic amyloid-β40 peptide initiates cell death after cultures were preconditioned with sublethal concentrations of buthionine sulfoximine (1.5 μM), a compound that enhances endogenous ROS production. The damage is predominantly in the granule cell layer of the dentate gyrus, with additional cell loss in pyramidal neurons. The dying neurons evoke reactive gliosis with altered (accentuated) TRPV4 expression. In this model, neuronal damage is attenuated by the universal TRP blocker, RR [162]. Of note, the PARP (poly(ADP-ribose)polymerase) inhibitor SB-750139 also attenuates cell death initiated by amyloid β-peptide in rat striatum neurons [163]. PARP (in concert with Ca^2 +) activates endogenous TRPM2 channels, a neuronal “redox sensor.” Thus, it is entirely possible that TRPV4 up-regulation is coincidental and the real target for ROS-mediated cell death is TRPM2. Last, hyperforin (a TRPC6 activator) blocks the formation of amyloid-β aggregates in vitro and decreases astrogliosis and microglia activation in vivo. Importantly, hyperforin improves spatial memory formation in animal models of AD [164]. Indirectly, these findings implicate TRPV4, TRPM2, and TRPC6 in the pathogenesis of AD. If these observations hold true in humans, combined pharmacological blockade of TRPM2 and TRPV4 and stimulation of TRPC6 may be beneficial in AD patients.

Autoimmune Encephalomyelitis and Multiple Sclerosis

Cholera toxin-B binds to the branched pentasaccharide moiety of the ganglioside, GM1. This protein-ganglioside interaction is instrumental in initiating the signal transduction pathway that is responsible for diarrhea. In murine experimental autoimmune encephalomyelitis (EAE), administration of the GM1 cross-linking unit of cholera toxin (the so-called B-toxin) is beneficial. Cholera toxin-B is a binding partner for the endogenous lectin, galectin-1. In these animals, both cholera toxin-B and galectin-1 caused symptomatic improvement. Conversely, mice lacking GM1 demonstrate enhanced susceptibility to EAE. Polyclonal activation of murine regulatory T (Treg) cells up-regulates galectin-1. Furthermore, activation of CD4 + and CD8 + effector T (Teff) cells elevates GM1. Of importance to the topic of this chapter, activation of Teff cells also up-regulates TRPC5, which, in turn, mediates the Ca^2 + influx on GM1 cross-linking by either galectin-1 or cholera toxin-B. Knockdown by shRNA of Trpc5 in Teff cells blocks the contact-dependent proliferation inhibition by Treg cells. These observations suggest a role for TRPC5 in the negative control (suppression) of autoimmunity [165].

Multiple sclerosis (MS) is a chronic, relentlessly progressive demyelinating disorder. Although the pathogenesis of MS is unknown, increased glutamate production is thought to play a role in the inflammation-driven neurodegenerative process. In support of this hypothesis, CSF collected from MS patients potentiates glutamate-mediated neuronal swelling through a mechanism that seems to involve both IL-1β signaling and increased AMPA-R stimulation. Indeed, IL-1β is significantly higher in the CSF of patients with active MS. It was hypothesized that TRPV1 is an essential mediator for the synaptic action of IL-1β on central glutamatergic synapses [166]. In keeping with this hypothesis, in a mouse model of EAE Trpv1 null animals show reduced infiltration of the CNS by autoreactive T-cells [167]. In MS patients, a missense SNP in the TRPV1 gene was correlated to the risk of progressive disease [167]. Taken together, these findings identify TRPV1 as a potential novel therapeutic target in MS patients.

Anxiety disorders are the most common form of mental illness in the United States, affecting one out of six Americans age 18 and older. In mice, several TRP channels (including TRPC4, TRPC5, and TRPV1) are expressed in brain areas implicated in the control of fear and anxiety. In behavioral experiments, constitutive ablation of Trpc4 or Trpc5 was associated with diminished innate fear and anxiety levels. Selective knockdown of Trpc4 in the lateral amygdala via lentiviral-mediated gene delivery of RNAi mimicked the behavioral phenotype of the Trpc4 knockout mouse. It was speculated that TRPC4 is a crucial downstream target for two G_αq11 protein-coupled signaling pathways, activated via Group-I mGluRs and CCK2 receptors, respectively [168]. These observations imply that TRPC4 and/or TRPC5 blockers may constitute a new class of anxiolytic drugs.

Several lines of experimental evidence implicate TRPV1 in anxiety and panic responses. In behavioral studies, TRPV1 activation by capsaicin (microinjected into the periaqueductal gray) initiates anxiety-like behavior, including less frequent entry into the open arm of the maze test, as well as reduced number of stretched-attend postures and head dippings. TRPV1 blockade by capsazepine did not change the behavior of the animals in the elevated maze test. However, when given before capsaicin, capsazepine completely blocked the anxiogenic-like effect. These findings imply that (1) TRPV1 activation in the periaqueductal gray causes anxiety-like behavior in the mouse, and (2) these TRPV1 receptors are not tonically active [169]. Other studies, however, are more consistent with a tonically active TRPV1. For example, capsazepine inhibits the escape response in the elevated T-maze test [170], and Trpv1 null animals exhibit diminished innate fear response and anxiety-like behavior [171]. In addition, it was shown that TRPV1 might be a functional tool to prevent the risks associated with the long-term use of benzodiazepines [77].

So, what is the endogenous substance that evokes anxiety by activating TRPV1? An interesting candidate molecule is anandamide. In the T-maze assay, microinjection of the selective FAAH inhibitor, URB 597, into the hippocampus produces anxiolytic-like effects, presumably by elevating endogenous anandamide levels. Further increase in anandamide levels, however, exerts the opposite effect. The TRPV1 antagonist AMG 9810 does not interfere with the anxiolytic effect of URB 597 but blocks the anxiogenic action. These findings suggest that the beneficial effect of anadamide (or URB 597) is mediated by CB1-Rs, whereas TRPV1 activation contributes to increased anxiety [172]. These findings add TRPV1 antagonists to the list of potential new anxiolytic agents.

Unfortunately, the TRPV1 literature is confusing with some studies suggesting that it is TRPV1 activation that improves mood and reduces anxiety. In mice, nicotine induces depression-like behavioral alterations, similar to those seen in other murine models of depression such as the repeated immobilization stress. In both the nicotine and immobilization stress models, capsaicin and olvanil administered intraperitoneally exhibited significant antidepressant-like activity. By contrast, anandamide and N-arachidonyldopamine (NADA) lack antidepressant-like effects. In accord, the antidepressant-like effect of capsaicin and olvanil was reversed by capsazepine, but not the CB1R antagonist, AM 251 [173]. These observations imply that TRPV1 activation may improve mood, especially in “cold turkeys” suffering from nicotine withdrawal symptoms.

Incensole acetate, the main active ingredient in Boswellia resin, activates TRPV3 in expression systems. In vivo, incensole acetate causes anxiolytic-like and antidepressive-like behavioral effects in wild-type, but not Trpv3 knockout, mice, with concomitant changes in c-Fos activation in the brain [174]. These findings imply a therapeutic potential for TRPV1 agonist in anxiety disorders and depression.

Schizophrenia

Schizophrenia is a disabling mental disorder that affects ~ 1% of the population worldwide. It runs in families (identical twins have a ~ 50% chance of developing the disease), suggesting a genetic predisposition for neurochemical malfunction in the brain. Disrupted in schizophrenia-1 (DISC1) is a protein implicated in schizophrenia (also in bipolar disorder, major depressive disorder, and autism). The function of DISC1 is to modulate cAMP signaling by increased cAMP catabolism. DISC1 disruption by shRNA knockdown increases intracellular Ca^2 + waves in response to mGluR activation. Furthermore, it decreases TRPC-mediated sustained depolarization. It was hypothesized that diminished DISC1 function disrupts the normal pattern of prefrontal cortex activity through the loss of cAMP regulation of mGluR-mediated intracellular Ca^2 + waves; this eventually leads to perturbations in TRPC channel activity [175]. As of today, it is unclear which member of the TRPC subfamily is involved in this response.

A link between TRPV1 and schizophrenia was postulated, but the experimental evidence is at best preliminary. In rats, capsaicin desensitization leads to behavioral changes (e.g., learning impairments in the novel object recognition test) that are somewhat reminiscent of those seen in patients with schizophrenia [176]. Furthermore, spontaneously hypertensive (SH) rats show schizophrenia-like deficits in social interactions that are ameliorated by atypical antipsychotics. Capsaicin (2.5 mg/kg) increases social interaction of in both SH and normotensive rats and decreases locomotion in SH rats [177]. These findings might be interpreted to imply that loss of TRPV1 function could contribute to the pathogenesis of schizophrenia, whereas TRPV1 activation may be beneficial in these patients.

Rett Syndrome

Rett syndrome is a neurodevelopmental disorder that affects intellectual ability. Children with Rett syndrome exhibit autism-like behavior combined with impaired motor functions. In the hippocampus, BDNF activates TRPC3 to increase neuronal dendritic spine density. Indeed, knockdown of Trpc3 prevents the increase in spine density caused by BDNF application. It was hypothesized that dysfunction in the BDNF-TRPC3 interaction may contribute to the pathomechanism of Rett syndrome by causing abnormal dendritic spine density [178]. This model is supported by findings in mice that lack the methyl-CpG-binding protein-2 (Mecp2), a model of Rett syndrome. In symptomatic Mecp2 mutant mice, membrane currents and dendritic Ca^2 + signals evoked by recombinant BDNF are impaired. In the hippocampus of Mecp2 mutants, both TRPC3 and TRPC6 are decreased. BDNF mRNA and protein levels are also lower in Mecp2 mutant hippocampus and dentate gyrus granule cells. Chromatin immunoprecipitation suggest that Trpc3 is a target of Mecp2 transcriptional regulation. According to these results, correction of the impaired BDNF-TRPC6 signaling is a potential therapeutic strategy in Rett syndrome [179].

Bipolar Disorder

Two indirect findings implicate TRPM2 in the pathogenesis of bipolar disorder. First, B-lymphoblast cell lines established from bipolar disorder patients when exposed to rotenone (a broad-spectrum, neurotoxic pesticide and a known activator of TRPM2) exhibit reduced cell viability compared to healthy controls [180]. Second, in case-control studies, a number of SNPs in the TRPM2 gene were reported to increase the risk for developing bipolar disorder. Interestingly, the C-T-A haplotype of SNPs rs1618355, rs933151, and rs749909 was significantly associated with early age at onset of the disease [181].

Two members of the canonical TRP subfamily, TRPC1 and TRPC6, have been linked to cocaine abuse and addiction. Conditional forebrain Trpc5 knockdown mice exhibit an increase in self-administration of cocaine without prior operant training. This observation was interpreted to imply a negative control of TRPC5 over addictive behavior [182]. Mice whose brain expresses the HIV-1 Tat protein show a heightened response to cocaine and appear to be vulnerable to relapse. This observation establishes a connection between HIV infection and drug abuse. Pretreatment of rat hippocampal neuronal progenitor cells with platelet-derived growth factor-BB (PDGF-BB) restores proliferation that had been impaired by HIV-1 Tat. TRPC1 appears to be a downstream target for PDGF-BB. These findings highlight TRPC1 as a novel target that regulates cell proliferation mediated by PDGF-BB with implications for therapeutic intervention in cocaine addiction [183].

Opioid Addiction

In the rat, TRPC6 is expressed in CSF-contacting neurons, with increased levels found during morphine dependence and withdrawal [184]. In mice, repeated morphine administration up-regulates TRPV1 expression in the dorsal striatum. TRPV1 agonists potentiate, whereas TRPV1 antagonists attenuate, morphine reward in the conditioned place preference paradigm. TRPV1 antagonist treatment also suppresses morphine-induced increases in μ-opioid receptor binding. Thus, brain TRPV1 may represent a novel therapeutic target to treat morphine-addictive disorders [185].

Intracerebral hemorrhage (hemorrhagic stroke) is a devastating event that stems from the rupture of blood vessels in the brain, with the subsequent accumulation of blood in the parenchyma. There is good evidence that blood-derived factors induce excessive inflammatory responses that, in turn, contribute to the progression of brain injury. When the blood-brain barrier is disrupted, thrombin leaks to the brain parenchyma to cause astrogliosis. Furthermore, thrombin up-regulates TRPC3. In mice, the TRPC3 blocker Pyr3 improves functional outcome and attenuates astrogliosis after hemorrhagic stroke. These findings highlight TRPC3 as a novel therapeutic target for the treatment of hemorrhagic brain injury [186].

Intracerebroventricular injection of hyperforin, a TRPC6 activator, ameliorates the brain damage that occurs after transient focal cerebral ischemia in rats. When applied immediately after middle cerebral artery (MCA) occlusion, hyperforin reduced the infarct volumes and increased neurologic scores at 24 hours after reperfusion. These beneficial effects were accompanied by elevated TRPC6 activity [187]. Resveratrol is another natural product that protects the brain from ischemia/reperfusion injury. In the transient MCA occlusion model, resveratrol protects neurons by inhibiting the proteolysis of TRPC6 by calpain. There are striking similarities between hyperforin and resveratrol actions, suggesting a shared biochemical pathway. For example, both hyperforin and resveratrol elevate TRPC6 and CREB (cAMP-response element binding protein) activities. When the MEK (MAPK/ERK kinase) or CaMK-IV activity was inhibited, the neuroprotective effect of resveratrol was lost. Taken together, these findings suggest that hyperforin and resveratrol protect the brain from ischemic injury through the TRPC6-MEK-CREB and TRPC6-CaMKIV-CREB pathways [188].

Excitotoxicity induced by NMDA receptor-mediated intracellular Ca^2 + overload is a major cause of delayed neuronal death after ischemic stroke. As we saw earlier, TRPC6 protects neurons from ischemic brain damage. For example, the infarct volume in Trpc6 transgenic mice is much smaller than that in wild-type littermates after MCA occlusion. The Trpc6 transgenic mice also had better behavior performance and lower mortality than the control animals. These observations imply that increasing TRPC6 activity could be a potential strategy for stroke prevention and therapy [189].

Chronic cerebral hypoperfusion is a risk factor for the development of vascular dementia. In men, vascular dementia accounts for 20-40% of all dementia cases. In mice, bilateral carotid artery occlusion is used to induce global chronic cerebral hypoperfusion. These animals show impairment in locomotion and motor coordination, as well as deficits in learning and memory formation. TRPV1 antagonists are protective against hypoperfusion-induced motor coordination impairment and cognitive decline [190]. Of note, following MCA occlusion the expression of TRPV1 is significantly increased in the hippocampus [191].

Inducing mild hypothermia is a promising therapeutic approach in stroke patients. In rodents, capsaicin evokes a rapidly developing and transient drop in body temperature. Rinvanil is a potent, synthetic capsaicin congener. Intraperitoneal rinvanil administration induces mild hypothermia and protects neurons from the ischemic damage that develops following transient MCA occlusion [192]. This observation highlights the therapeutic potential of TRPV1-mediated hypothermia to minimize brain damage in stroke patients.

Incensole acetate protects against the neurological deficit caused by head trauma. In mice, incensole acetate also attenuates ischemic neuronal damage and reperfusion injury by limiting neuro-inflammation. The protective effects of incensole acetate were absent in Trpv3 deficient mice and were reversed by TRPV3 inhibitors [193].

In adult rat cortical and hippocampal astrocytes, TRPV4 expression is markedly increased 7 days after hypoxia-ischemia insult. The increase in TRPV4 expression coincides with the development of astrogliosis (astrogliosis is an abnormal astrocytic proliferation that occurs in response to neuronal death). In brain slices or cultured hippocampal astrocytes, the TRPV4 agonist 4αPDD elevates Ca^2 + and activates a cationic current that is prevented by the universal TRP channel antagonist, RR. Importantly, hypoxic-ischemic injury augments the responses of astrocytes to 4αPDD. These observations imply a role for TRPV4 in the development of astrogliosis that follows ischemic insults [194]. Of note, microglial cells also express TRPM2 with increased levels following ischemic brain injury [195].

In addition to astrocytes, TRPV4 is expressed in hippocampal CA1 pyramidal neurons. These neurons are activated by both 4αPDD and hyposmotic insult; these responses were prevented by the TRPV4 antagonist, HC-067047, and the NMDA-R antagonist, AP-5, indicating that TRPV4 activation potentiates NMDA-R response. When given 60 minutes after MCA occlusion, HC-067047 reduced the size of the cerebral infarct. These findings indicate that activation of TRPV4 increases NMDA-R function, which in turn potentiates glutamate excitotoxicity. If this hypothesis holds true, TRPV4 antagonists may exert potent neuroprotection against cerebral ischemia injury [196].

In stroke models, TRPM2 shows a very unusual behavior. TRPM2 expression is similar in male and female control rats. During reperfusion following in vitro ischemia, TRPM2 channels get activated only in neurons of male animals [197]. In keeping with this finding, shRNA-mediated knockdown of Trpm2 expression protected male, but not female, neurons following in vitro oxygen-glucose deprivation. Importantly, the TRPM2 inhibitor, clotrimazole, reduced infarct volume in male mice, while having no effect in female animals [198]. If these observations hold true in humans, TRPM2 represents a potential target for protection against cerebral ischemia in male stroke patients.

Traumatic Brain Injury

Traumatic brain injury initiates a cascade of complex biochemical changes, including oxidative stress, edema, inflammation, and excitotoxicity. Deferoxamine is a chelator that attenuates Fe^2 +-induced toxicity in rats with traumatic brain injury. These animals show a significant increase in brain Fe^2 + on day 28, accompanied by a corresponding elevation in TRPC6 levels. Deferoxamine ameliorated the deficits in spatial learning and memory, supporting the notion that deferoxamine may reduce brain injury accentuated by Fe^2 + overload by interfering with a biochemical pathway that involves TRPC6 [199]. In an impact-acceleration model of diffuse traumatic brain injury in rats, TRPM2 mRNA and protein levels are elevated in the cerebral cortex [200].

Microvascular failure exacerbates the damage caused by traumatic brain injury via a combination of cerebral edema formation and progressive secondary hemorrhage. In rat models of traumatic brain injury, two transport proteins have been identified in brain endothelial cells as critical mediators of edema formation: the constitutively expressed Na⁺-K⁺-Cl⁻ cotransporter, NKCC1, and the trauma/ischemia-induced SUR1/TRPM4 channel. Whereas NKCC1 function requires ATP, activation of SUR1/TRPM4 occurs only after ATP is depleted. With critical ATP depletion, sustained opening of SUR1/TRPM4 channels may result in the oncotic death of endothelial cells, leading to capillary fragmentation and progressive secondary hemorrhage. Bumetanide and glibenclamide are antidiabetic drugs that inhibit NKCC1 and the SUR1/TRPM4 channel, respectively. In animal models of traumatic brain injury, bumetanide and glibenclamide protects neurons by preventing injury-associated capillary failure [201–204]. Following spinal cord injury, Trpm4 mRNA and protein are up-regulated in capillaries, preceding their fragmentation and formation of petechial hemorrhages. Trpm4 gene suppression (by antisense) or deletion (Trpm4 knockout mice) preserved capillary structural integrity and eliminated secondary hemorrhage. In these TRPM4-deficient animals the lesion volume was reduced and the neurological function was improved compared to controls [205]. In cerebral arteries, TRPM4 might be a regulator of the myogenic constrictor response that occurs in response to increases in intravascular pressure, the so-called Bayliss effect. In rats, suppression of cerebrovascular TRPM4 expression by antisense oligonucleotides reduces myogenic constriction by 70% to 85% [206].

In mice, suppressing the expression of Trpm7 in hippocampal CA1 neurons confers resistance to ischemic cell death [207]. In these animals, hypoxia increases Mg^2 + via TRPM7 in the hippocampus, and this contributes to cell death [208]. In men, conversely, depletion of intracellular Mg^2 + is known to occur after stroke when it heralds poor neurological outcome. TRPM7 and TRPM6 play important roles in regulating Mg^2 + homeostasis; whether or not these channels contribute to neuronal injury remains controversial [209]. To determine whether TRPM7 gene variants may increase (or decrease) the risk of ischemic stroke, a large cohort (14 916) of healthy American men were genotyped for 16 SNPs in the TRPM7 gene. Of these men, 245 subsequently suffered ischemic stroke. All SNPs were in Hardy-Weinberg equilibrium. Overall allele, genotype, and haplotype distributions were similar between cases and controls. Furthermore, marker-by-marker conditional logistic-regression analysis adjusted for potential risk factors showed no evidence for an association between any of the SNPs tested and ischemic stroke [210].