Mice have been generated with inactivating mutations in the Tsc1 or Tsc2 genes [27, 28]. Homozygosity for these mutations results in embryonic lethality [27, 28], but heterozygous mutants are viable and have been assessed with regard to neurological phenotypes, as discussed below.

Goorden et al. assessed behavior and cognition in Tsc1 ^+/− mice. These animals showed learning and memory impairments, as revealed by experiments using the hidden version of the Morris water maze task [29]. Escape latencies were normal in the mutants, but Tsc1 ^+/− mice searched less selectively (quadrant occupancy and target quadrant measures) for the escape platform during a probe trial that was given after completion of training [29]. Additionally, Tsc1 ^+/− mice showed reduced freezing levels during a context test in a context fear conditioning paradigm [29]. Freezing in the mutants was unaltered when a tone was used to signal the shock (cued fear conditioning) [29]. These findings are consistent with hippocampus-dependent learning and memory impairments in Tsc1 haploinsufficient mice.

Additional behavioral testing revealed social interaction abnormalities in Tsc1 ^+/− mice: The mutants spent significantly less time exploring a conspecific in a social interaction paradigm [29]. Reductions in nest building behavior were also found in this model [29]. Importantly, Tsc1 ^+/− mice do not display obvious seizures [29]. Brain MRI and histological assessment revealed no obvious brain pathology in these animals (no tuber-like pathology, normal neuronal soma size; normal numbers of primary and secondary dendrites on hippocampal granule neurons; normal spine density on hippocampal granule neurons) [29]. Behavioral impairments in Tsc1 ^+/− mice, therefore, emerged in the absence of obvious brain pathology and seizures and, hence, other factors are required to explain the behavioral alterations in this model.

Ehninger et al. performed a detailed assessment of cognitive function and behavior in heterozygous Tsc2 mutant mice [30]. Tsc2 ^+/− mice showed learning and memory impairments on three hippocampus-dependent tasks [30]. First, during the probe trial in a hidden version of the Morris water maze task, they showed less selective searching (quadrant occupancy, target crossings) than wild-type littermate controls, suggesting that these animals had not learned the task as well as their wild-type counterparts [30]. Secondly, Tsc2 ^+/− mice made more across-phase errors on a delayed-non-match-to-version of an eight-arm radial maze that was used to test spatial working memory in the mutants [30]. Across-phase errors (as opposed to within-phase errors that were not different between the mutants and controls) are particularly sensitive to hippocampal dysfunction in experimental animals [31, 32]. The Tsc2 ^+/− mice also displayed context discrimination impairments in a contextual fear conditioning paradigm, that is they showed similar levels of freezing to the training context (in which they were conditioned) and a novel unrelated context [30], indicating that conditioned responses were not preferentially expressed to the training context, as was the case in wild-type littermate controls.

Additional behavioral testing showed that motor coordination, social interaction, anxiety-related behaviors and exploratory behavior were unaltered in Tsc2 ^+/− mice [30]. Like Tsc1 ^+/− mice, heterozygous Tsc2 mutants also lacked obvious structural brain abnormalities and behavioral seizures. Ehninger et al. also studied long-term potentiation (LTP), a form of hippocampal synaptic plasticity, at the Schaffer Collateral-CA1 synapse in the mutants [30], because activity-dependent synaptic modifications are thought to play an important role in hippocampus-dependent memory processes. Tsc2 ^+/− mice showed a lowered threshold for the induction of late-phase long-term potentiation (LTP) in the hippocampus [30], which could conceivably perturb certain forms of learning and contribute to learning and memory impairments in Tsc2 ^+/− mice. Brief treatment of adult Tsc2 ^+/− mice with the mTOR inhibitor rapamycin restored learning and memory impairments in this mouse model [30], suggesting that disinhibited mTOR signaling in the mature brain contributes to TSC-related cognitive phenotypes. Rapamycin treatment also restored abnormal late-phase LTP in the Tsc2 ^+/− hippocampus to levels that corresponded roughly to those of controls [30]. These findings suggest that at least some aspects of the cognitive deficits in TSC, such as memory difficulties, may be caused by functional defects in the adult brain, indicating that the may be accessible to treatment, even in adulthood.

Separation calls in the ultrasonic range were also assessed in Tsc2 ^+/− mice. Tsc2 ^+/− pups showed vocalization rates that were indistinguishable from those of wild-type (WT) littermates [33, 34]. Young et al. report that pups of both genotypes (Tsc2 ^+/−, WT) showed higher numbers of vocalizations when they were born to Tsc2 ^+/− dams than when they were born to WT dams [33]. Separation from the dam after temporary reunion typically induces elevated levels of vocalizations by the pups (maternal potentiation). Maternal potentiation was observed in Tsc2 ^+/− and WT pups born to WT mothers and in Tsc2 ^+/− pups born to Tsc2 ^+/− mothers, but not in WT pups born to Tsc2 ^+/− mothers [33], suggesting an interactive effect of dam and pup genotypes in modulating maternal potentiation behavior.

Both, Tsc1 ^+/− and Tsc2 ^+/− mice were assessed in different versions of social interaction paradigms. While Tsc2 ^+/− mice showed no obvious alterations in social approach behavior as measured in the three-compartment apparatus [30], impairments in social approach behavior could be triggered in Tsc2 ^+/− mutants by combining the Tsc2 mutation with a gestational immune activation paradigm [35]. In another study, using a social behavioral paradigm that employed unrestrained pairs of mice, Tsc2 ^+/− mice were reported to display reduced engagement in social interactions [36]. Similar observations were reported for Tsc1 ^+/− mice in this study [36], a finding that is in line with prior observations in the Tsc1 ^+/− model [29]. A 2-day treatment with rapamycin restored reduced social interaction behavior in adult Tsc1 ^+/− and Tsc2 ^+/− mice, while this treatment had no effect on the behavior in wild-type controls [36].

Auerbach et al. contrasted the effects of a heterozygous Tsc2 mutation with those that an Fmr1 mutation (model of Fragile X syndrome) has on protein synthesis, hippocampal synaptic plasticity and behavior [37]. Using hippocampal slices prepared from young (P25–35) Tsc2 ^+/− mice and controls, they find reduced metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) in the mutants (i.e., LTD induced by either the group I mGluR agonist (S)-3,5-Dihydroxyphenylglycine (DHPG) or induced by patterned electrical stimulation) [37]. Metabolic labeling experiments showed a surprising reduction of protein synthesis levels in Tsc2 ^+/− hippocampal slices, both globally and with respect to the translation of specific proteins that play a role in LTD (i.e., Arc) [37]. Rapamycin recovered mGluR-LTD in the mutants and led to an increase in protein synthesis in Tsc2 ^+/− hippocampal slices [37]. Furthermore, the mGluR5-positive allosteric modulator [3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide] (CDPPB) was shown to restore mGluR-LTD in Tsc2 ^+/− slices and increased global protein synthesis, as well as translational expression of Arc [37]; a single injection of CDPPB prior to context fear conditioning improved context discrimination impairments in Tsc2 ^+/− mice, while it had no effect on wild-type controls [37]. These findings indicate that synaptic, as well as behavioral phenotypes of Tsc2 ^+/− mice can be corrected by acutely activating mGluR5 signaling in the adult animal, which is in contrast to findings in animal models of Fragile X syndrome where inhibition of mGluR5 signaling affords phenotypic improvements [38]. Indeed, crossing the Tsc2 ^+/− mutation into an Fmr1 ^−/y background restored mGluR-LTD impairments in Tsc2 ^+/− hippocampal slices and rescued context discrimination impairments in Tsc2 ^+/− mice [37]. These findings, therefore, support the hypothesis that altered neuronal translational regulation contributes to the pathophysiology of different autism-associated syndromes [39], but indicate that the directionality of effects may differ between syndromes.

Similar to the findings discussed above [37], another recent study also reported hipocampal mGluR-LTD (induced by group I mGluR agonist DHPG) impairments in slices generated from young (21 days old) Tsc2 ^+/− animals, which were also amenable to correction by rapamycin [40]. Potter et al., however, also report that hippocampal mGluR-LTD was not different between Tsc2 ^+/− and wild type when they looked at slices generated from adult animals [40]. Nevertheless, in contrast to slices from adult wild-type mice, mGluR-LTD in Tsc2 ^+/− slices was not sensitive to rapamycin [40], which may have been related to increased mGluR5 expression and Erk signaling in the mutants. This idea is supported by the observation that rapamycin-sensitivity of mGluR-LTD in Tsc2 ^+/− slices could be restored by inhibiting mGluR5 or Erk signaling [40].

Potter et al. also observed that Tsc2 ^+/− slices developed higher levels of epileptiform bursting activity than wild-type slices when incubated with the group I mGluR agonist DHPG [40]. Epileptiform activity could be reduced in the mutants by pharmacological inhibition of Erk or mGluR5 signaling [40], indicating that mGluR5/Erk play a role in DHPG-induced hyperexcitability in Tsc2 ^+/− hippocampus. They also tested whether the inverse mGluR5 inverse agonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) restores behavioral changes in Tsc2 ^+/− mice. They report that a short treatment with MPEP improves perseverative behaviors of adult Tsc2 ^+/− mice on a radial arm water maze [40]. Together these data indicate that modulation of mGluR5 signaling has therapeutic effects on hyperexcitability and perseverative behavior in this TSC mouse model. It remains to be clarified under which conditions a positive (CDPPB; [37]; see above) vs. a negative (MPEP; [40]) modulation of mGluR5 signaling is beneficial in Tsc2 ^+/− mice.

Another mouse model of TSC was engineered by expressing a dominant negative Tsc2 transgene using a Cytomegalovirus (CMV) promoter (dominant negative Tsc2 transgenic mice; Tsc2-DN mice) [41, 42]. This modified Tsc2 transgene carries mutations affecting two structural motifs of the TSC2 protein; deletion of amino acid residues 1,617 through 1,655 should disrupt the structural integrity of the GAP domain on tuberin; additionally, amino acid residues 1,679 through 1,742 were substituted, which renders the rabaptin-5 domain of tuberin nonfunctional. These genetic modifications are then thought to interfere with the normal GAP activitity of tuberin and its rabaptin-5 binding by competing with and displacing endogenous TSC2 protein.

Chevere-Torres et al. performed hippocampal slice physiological studies in this model, which showed normal basal synaptic transmission at the Schaffer collerateral-CA1 synapse, normal paired-pulse facilitation, as well as normal LTP (E-LTP, L-LTP) [43]. Similar to the findings in Tsc2 ^+/− mice [37, 40], Tsc2-DN mice showed impairments in mGluR-LTD (slices from 4- to 6-week-old animals were examined) while displaying normal NMDAR-dependent LTD (induced by low-frequency stimulation; this form of LTD is thought to be protein synthesis-independent) [43]. mGluR-LTD impairments in Tsc2-DN mice were similar in magnitude to mGluR-LTD impairment in animals with either a conditional heterozygous deletion of Tsc1 targeted to neurons or a homozygous neuronal deletion of Tsc2 (in both cases, using animals expressing Cre recombinase from an αCaMKII promoter) [43], showing that LTD deficits are caused by both, the inactivating mutations as well as the dominant negative transgene, and demonstrating that the LTD impairments in the conditional mutants are due to cell-autonomous effects in neurons.

Further experiments on hippocampal tissue from the Tsc2-DN mice showed that the phosphorylation of ribosomal protein S6 at the mTORC1-dependent phosphorylation sites Ser240/244 did not differ between mutants and controls, while phosphorylation at the Ser235/236 sites (which may be due to mTORC1 or Erk) was significantly increased [43]. In line with these results, there was a significant increase of ERK phosphorylation in Tsc2-DN mice [43], which might be caused by the overexpression of TSC2 in this model [43] followed by Rheb-dependent disinhibition of ERK signaling [44]. Inhibition of abnormally elevated Erk phosphorylation using the Mek inhibitor U0126 restored mGluR-LTD in Tsc2-Dn hippocampal slices [43], suggesting that elevated Erk signaling played a role in altered mGluR-LTD in this model. The conditional Tsc1/2 mutants mentioned above, however, did not show increased Erk phosphorylation [43], indicating that mGluR-LTD deficits in different TSC models may not necessarily involve the same mechanistic underpinnings.

Ehninger and Silva performed a behavioral assessment of Tsc2-Dn mice [45]. These studies revealed increased levels of anxiety-related behaviors in these mutants [45]: On the elevated plus maze, the mutants showed decreased amounts of time spent on the open arms and there was a trend towards decreased center time in the open field. These findings are of interest in light of the elevated rates of anxiety disorders associated with TSC in humans [8, 9, 13, 46, 47] and suggest a biological foundation for this association. There were only mild effects on hippocampus-dependent learning and memory (spatial learning and memory in the Morris water maze; context discrimination) in this model [45].

Behavioral studies by Chevere-Torres et al. showed abnormalities in social behaviors in Tsc2-Dn mice [48]. Experiments using a three-compartment chamber demonstrated normal social approach behavior in the mutants, but showed an impaired preference for social novelty in Tsc2-Dn mice [48]. The mutants also showed reduced interaction scores in a reciprocal social interaction task (in the context of which the object mouse is not restrained but able to move freely) [48]. Further behavioral studies showed impaired motor coordination in the mutants [48]. Cerebellar dysfunction could potentially contribute to both motor coordination impairments and social behavioral deficits in TSC models [49]. Indeed, evidence is available that suggests that cerebellar pathology is present in Tsc2-Dn mice [41]. Sensorimotor gating (pre-pulse inhibition) and repetitive behaviors (marble burying, self-grooming) were reported to be normal in the transgenic mice [48].

The heterozygous TSC mouse models and the dominant-negative transgenic model described above show a number of biochemical changes, neurophysiological alterations and behavioral deficits with relevance for the disease. Other aspects of TSC are, however, not captured in these models, including neuropathological findings and seizures. For these reasons, a number of additional models have been developed that all involve restricted homozygous TSC gene deletions in mice.

Homozygous deletion of Tsc1 in neurons, using either a SynI (permitting recombination of the floxed allele in excitatory and inhibitory neurons) or αCaMKII promoter (recombination starting postnatally and targeted primarily to excitatory neurons) to drive Cre recombinase, resulted in mice showing poor weight gain and severely compromised survival [30, 50, 51]. Animals surviving past the first few weeks of life showed severe neurological impairments, including pronounced hypoactivity, tremor, kyphosis, pathological hindlimb clasping, and an aberrant tail position [30, 50, 51].

Neuronal Tsc1 mutants showed an overall preserved lamination of cortex and hippocampus and no focal tuber-like pathology [30, 50, 52]. However, neuronal cell enlargement and dysplastic features of neurons, such as accumulation of nonphosphorylated neurofilaments and abnormal dendrite orientation, were reported [50, 52]. In αCaMKII-neuronal Tsc1 mutant mice, cell hypertrophy was accompanied by substantial brain enlargement [30]. In addition to morphological changes in neurons, these models also featured non-cell-autonomous glial alterations, such as severe myelination defects in SynI-neuronal Tsc1 mutants [50] and pronounced reactive astrogliosis in αCaMKII-neuronal Tsc1 mutant mice [30].

Spontaneous or handling-induced seizures were observed in a subset of αCaMKII-neuronal [53], as well as SynI-neuronal [50, 52] Tsc1 mutants. Furthermore, αCaMKII-neuronal Tsc1 mutants showed increased seizure severity following seizure induction with kainic acid [53]. Electrophysiological studies, performed on slices from SynI-neuronal Tsc1 mutants, revealed increased excitability (long-duration poly-spike responses in extracellular field recordings, increased burst duration during patch clamp recordings) under conditions of γ-aminobutyric acid A receptor blockade (bath application of bicuculline) [52]. Addition of an AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor antagonist abolished bursts and intrinsic excitability was normal [52], indicating that abnormal electric activity may have been generated synaptically.

A recent paper described the development of an allelic series of mice with graded reduction of Tsc2 expression in neurons [54]. For this purpose, animals with a homozygous conditional hypomorphic allele (Tsc2 ^{c-del3/c-del3} SynI-Cre⁺) were generated, in addition to mice combining a heterozygous null allele with one conditional hypomorphic allele (Tsc2 ^k/c-del3 SynI-Cre⁺). It was estimated that the level of Tsc2 expression in neurons was reduced to 13 % of the normal levels in Tsc2 ^{c-del3/c-del3} SynI-Cre⁺ mice and to 7 % of normal in Tsc2 ^k/c-del3 SynI-Cre⁺ mice [54], demonstrating a substantial reduction of Tsc2 expression, even from the hypomorphic allele. Not unexpectedly, both lines showed strong activation of the mTORC1 pathway and many of the typical features that had been previously described for conditional neuron-specific homozygous Tsc1 mutants [54] (see above). These phenotypes included compromised survival, poor postnatal weight gain, brain enlargement, enlargement of neuronal soma size in the neocortex, neurological and behavioral alterations, such as hunchback, hindlimb clasping, and pronounced hyperactivity in the open field assay [54]. Generally, phenotypes were similar in Tsc2 ^{c-del3/c-del3} SynI-Cre⁺ and Tsc2 ^k/c-del3 SynI-Cre⁺ mice, but tended to be more severe in the model with lower Tsc2 expression (Tsc2 ^k/c-del3 SynI-Cre⁺). Social approach behavior was assessed in the three-compartment chamber; these experiments revealed normal social approach behavior in the mutants but impairments in the preference for social novelty in Tsc2 ^k/c-del3 SynI-Cre⁺ mice [54]. Learning and memory was assessed in a water T maze task in these animals. These studies revealed impaired acquisition and reversal learning in the mutants [54]. Taken together, these data are consistent with previous findings in heterozygous TSC models and conditional neuron-specific TSC models and show that, not surprisingly, a more severe loss of Tsc2 protein results in more severe disinhibition of mTORC1 signaling, more pronounced histopathological changes and a more adverse neurological outcome.

Bateup et al. homozygously deleted Tsc1 in hippocampal area CA1 by delivering adeno associated virus (AAV) expressing Cre recombinase into the hippocampi of floxed Tsc1 mice (age: P14–16 at the time of injection; experiments were performed 10–14 days later; EGFP was also expressed from the viral vector) [55]. Two-photon in vivo imaging analyses showed no obvious alterations in spine morphological measures [55], which is in contrast to observations made in organotypic slices when a similar approach was used to delete Tsc1 [56]. Consistent with findings in other TSC mouse models [37, 43], they report impaired mGluR-dependent LTD in Tsc1 mutant slices, using either a pharmacological (i.e., DHPG) or an electrical (i.e., PP-LFS in the presence of NMDAR antagonists) LTD induction protocol. NMDAR-dependent LTD, in contrast, which does not dependent on protein synthesis, was unaffected in Tsc1 mutants [55].

Using a similar approach, Bateup et al. assessed possible mechanisms for network hyperexcitability in homozygous Tsc1 mutant slices [53]. First, current clamp recordings from CA1 hippocampal neurons (injecting depolarizing currents to evoke action potentials) in the presence of synaptic blockers suggested reduced intrinsic neuronal excitability in Tsc1 KO neurons, as judged by an increased latency to the first spike and an increased action potential threshold [53]; this reduced excitability to current injection was likely related to an increased capacitance and decreased membrane resistance, respectively; the findings indicate that a cell-autonomous increase in intrinsic neuronal excitability does not account for the network hyperexcitability phenotype in Tsc1 mutants.

They also tested if altered glutamatergic neurotransmission may account for the hyperexcitability phenotype in homozygous Tsc1 mutant slices. Towards this end, simultaneous current clamp recordings of neighboring pairs of Tsc1 KO neurons and control neurons were performed, while the Schaffer collaterals were stimulated. These experiments showed no significant difference in the amplitude of evoked excitatory postsynaptic potentials (EPSP) in mutants and controls [53], suggesting that an enhancement of excitatory synaptic transmission did not account for the network hyperexcitability phenotype.

To test if changes in the inhibitory system may account for the network hyperexcitability phenotype in Tsc1 mutant slices, whole-cell recordings were performed on CA1 pyramidal cells that showed reduced mIPSC amplitudes in Tsc1 KO neurons [53], suggesting reduced numbers of postsynaptic GABA receptor abundance. Furthermore, paired recordings from neighboring mutant and control cells while stimulating Schaffer collaterals in the presence of glutamate receptor blockers showed reduced amplitudes of evoked inhibitory postsynaptic currents (IPSC) in Tsc1 KO neurons [53]. The data indicate that the loss of Tsc1 in CA1 neurons caused a cell-autonomous weakening of inhibition, while excitation was unaffected, resulting in an increased E/I ratio. Notably altered E/I ratios were observed previously in animal models of other syndromes associated with autism, such as Fragile X syndromes, suggesting potential shared pathophysiological mechanisms of distinct autism-related disorders [57]. Treating animals with rapamycin for 7 days prior to performing electrophysiological recordings normalized the E/I balance in Tsc1 mutants [53].

Abs et al. [58] examined the effects of an adult-onset Tsc1 deletion by crossing floxed Tsc1 mice to animals bearing a Cag-CreERT trangene [59], which allows inducible Cre translocation into the nucleus upon tamoxifen injection with subsequent recombination of the floxed Tsc1 allele. Short-term tamoxifen injection (2 days) in adult mutant mice caused homozygous deletion of Tsc1 in neural and non-neural cells and was lethal with a median survival of 8 days following the injections [58]. Prior to death, mutant mice developed seizures and interictal EEG abnormalities [58]. At 4 days after tamoxifen injection strongly p-S6-positive neurons were observed (excitatory and inhibitory), indicative of mTORC1 activation in neuronal cells [58]. Astrogliosis and hypomyelination, which are seen in neuron-specific Tsc1 KO mice [30, 50], were not observed at this time point [58], suggesting that these pathologies did not play a critical role in the seizure phenotype observed in this model. Hippocampal field recordings showed no evidence for alterations in basal synaptic transmission [58]. Inducible Tsc1 mutant mice showed a reduced threshold for the induction of hippocampal late-phase LTP [58], which is similar to the findings in heterozygous Tsc2 mutant mice [30]. Whole-cell recordings showed a higher number of action potentials upon depolarizing current injection in inducible Tsc1 mutant neurons compared to controls [58]. Moreover, there was a lower voltage threshold to elicit action potentials in inducible Tsc1 mutant neurons [58]. Neuronal hyperexcitability may, therefore, contribute to epileptogenesis in this model. Rapamycin treatment after seizure onset abolished seizures in this model [58], which is consistent with prior data that also showed that rapamycin is effective not only in preventing seizures, but can also ameliorate established seizure phenotypes [60].

The effects of conditional Tsc gene deletion in Purkinje cells was assessed in three studies published to date [49, 61, 62]. Reith et al. used a floxed Tsc2 allele combined with a Pcp2-Cre line to conditionally delete Tsc2 in cerebellar Purkinje cells [61]. This resulted in disinhibited mTORC1 signaling, ER and oxidative stress in Purkinje cells, apoptotic cell death and progressive loss of Purkinje neurons associated with gait abnormalities and impaired motor coordination [61]. Rapamycin treatment rescued Purkinje cell loss and gait ataxia in this model [61]. The authors also report decreased Purkinje cell density in postmortem samples of human TSC individuals [61], raising the possibility that Purkinje cell loss may also be a feature of the disorder in humans, although it cannot be ruled out at present that this observation is a secondary effect of seizures and/or antiepileptic medication.

Tsai et al. investigated the effects of conditional Tsc1 deletion in Purkinje cells on murine behaviors relevant to behavioral core features of autism spectrum disorders [49]. These studies were motivated by several links that exist between the cerebellar pathology and autism: Postmortem studies in ASD individuals showed reduced Purkinje neuron numbers [63]. Studies in TSC individuals show cerebellar abnormalities [64] correlated with increased autism-related symptomatology [65–67].

Both heterozygous and homozygous conditional Tsc1 deletion caused disinhibition of mTORC1 signaling in Purkinje cells, as evidenced by elevated p-S6 levels in these cells. Homozygous conditional deletion of Tsc1 resulted in Purkinje cell degeneration associated with progressive ataxia [49]. Heterozygotes, in contrast, showed no cell loss and motor impairments. Heterozygous and homozygous conditional Tsc1 deletion in cerebellar Purkinje cells resulted in a number of behavioral impairments relevant to behavioral core features of autism spectrum disorders [49]. Specifically, these mice showed impaired social approach behavior in a three-compartment assay, deficient reversal learning on a water T-maze task, and excessive grooming behavior. Mutant pups (heterozygous and homozygous) emitted more ultrasonic vocalizations when separated from their mother than controls. Electrophysiological studies revealed that synaptic input onto cerebellar Purkinje cells was unaltered in the mutants, but loss of Tsc1 in Purkinje neurons caused reduced intrinsic excitability of these cells [49]. Purkinje cells functionally couple via the deep cerebellar nuclei to a number of brain areas and disrupted Purkinje cell function could thereby lead to widespread perturbations in network functions. Neuropathological and behavioral alterations in these models depended on disinhibited mTOR signaling as treatment with the mTOR inhibitor rapamycin, starting at P7, prevented these phenotypes [49].