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Patients with diabetes often develop gastrointestinal motor problems, including gastroparesis. Previous studies have suggested this gastric motor disorder was a consequence of an enteric neuropathy. Disruptions in interstitial cells of Cajal (ICC) have also been reported. A thorough examination of functional changes in gastric motor activity during diabetes has not yet been performed. We comprehensively examined the gastric antrums of Lepob mice using functional, morphological, and molecular techniques to determine the pathophysiological consequences in this type 2 diabetic animal model. Video analysis and isometric force measurements revealed higher frequency and less robust antral contractions in Lepob mice compared with controls. Electrical pacemaker activity was reduced in amplitude and increased in frequency. Populations of enteric neurons, ICC, and platelet-derived growth factor receptor α+ cells were unchanged. Analysis of components of the prostaglandin pathway revealed upregulation of multiple enzymes and receptors. Prostaglandin-endoperoxide synthase-2 inhibition increased slow wave amplitudes and reduced frequency of diabetic antrums. In conclusion, gastric pacemaker and contractile activity is disordered in type 2 diabetic mice, and this appears to be a consequence of excessive prostaglandin signaling. Inhibition of prostaglandin synthesis may provide a novel treatment for diabetic gastric motility disorders.

KEY POINTS Enteric neurotransmission is essential for gastrointestinal (GI) motility, although the cells and conductances responsible for post-junctional responses are controversial. The calcium-activated chloride conductance (CaCC), anoctamin-1 (Ano1), was expressed by intramuscular interstitial cells of Cajal (ICC-IM) in proximal stomach and not resolved in smooth muscle cells (SMCs). Cholinergic nerve fibres were closely apposed to ICC-IM. Conductances activated by cholinergic stimulation in isolated ICC-IM and SMCs were determined. A CaCC was activated by carbachol in ICC-IM and a non-selective cation conductance in SMCs. Responses to cholinergic nerve stimulation were studied. Excitatory junction potentials (EJPs) and mechanical responses were evoked in wild-type mice but absent or greatly reduced with knockout/down of Ano1. Drugs that block Ano1 inhibited the conductance activated by carbachol in ICC-IM and EJPs and mechanical responses in tissues. The data of the present study suggest that electrical and mechanical responses to cholinergic nerve stimulation are mediated by Ano1 expressed in ICC-IM and not SMCs. ABSTRACT Enteric motor neurotransmission is essential for normal gastrointestinal (GI) motility. Controversy exists regarding the cells and ionic conductance(s) that mediate post-junctional neuroeffector responses to motor neurotransmitters. Isolated intramuscular ICC (ICC-IM) and smooth muscle cells (SMCs) from murine fundus muscles were used to determine the conductances activated by carbachol (CCh) in each cell type. The calcium-activated chloride conductance (CaCC), anoctamin-1 (Ano1) is expressed by ICC-IM but not resolved in SMCs, and CCh activated a Cl- conductance in ICC-IM and a non-selective cation conductance in SMCs. We also studied responses to nerve stimulation using electrical-field stimulation (EFS) of intact fundus muscles from wild-type and Ano1 knockout mice. EFS activated excitatory junction potentials (EJPs) in wild-type mice, although EJPs were absent in mice with congenital deactivation of Ano1 and greatly reduced in animals in which the CaCC-Ano1 was knocked down using Cre/loxP technology. Contractions to cholinergic nerve stimulation were also greatly reduced in Ano1 knockouts. SMCs cells also have receptors and ion channels activated by muscarinic agonists. Blocking acetylcholine esterase with neostigmine revealed a slow depolarization that developed after EJPs in wild-type mice. This depolarization was still apparent in mice with genetic deactivation of Ano1. Pharmacological blockers of Ano1 also inhibited EJPs and contractile responses to muscarinic stimulation in fundus muscles. The data of the present study are consistent with the hypothesis that ACh released from motor nerves binds muscarinic receptors on ICC-IM with preference and activates Ano1. If metabolism of acetylcholine is inhibited, ACh overflows and binds to extrajunctional receptors on SMCs, eliciting a slower depolarization response.

KEY POINTS: Electrical pacemaking in gastrointestinal muscles is generated by specialized interstitial cells of Cajal that produce the patterns of contractions required for peristalsis and segmentation in the gut. The calcium‐activated chloride conductance anoctamin‐1 (Ano1) has been shown to be responsible for the generation of pacemaker activity in GI muscles, but this conclusion is established from studies of juvenile animals in which effects of reduced Ano1 on gastric emptying and motor patterns could not be evaluated. Knocking down Ano1 expression using Cre/LoxP technology caused dramatic changes in in gastric motor activity, with disrupted slow waves, abnormal phasic contractions and delayed gastric emptying; modest changes were noted in the small intestine. Comparison of the effects of Ano1 antagonists on muscles from juvenile and adult small intestinal muscles suggests that conductances in addition to Ano1 may develop with age and contribute to pacemaker activity. ABSTRACT: Interstitial cells of Cajal (ICC) generate slow waves and transduce neurotransmitter signals in the gastrointestinal (GI) tract, facilitating normal motility patterns. ICC express a Ca(2+)‐activated Cl(−) conductance (CaCC), and constitutive knockout of the channel protein anoctamin‐1 leads to loss of slow waves in gastric and intestinal muscles. These knockout experiments were performed on juvenile mice. However, additional experiments demonstrated significant differences in the sensitivity of gastric and intestinal muscles to antagonists of anoctamin‐1 channels. Furthermore, the significance of anoctamin‐1 and the electrical and mechanical behaviours facilitated by this conductance have not been evaluated on the motor behaviours of adult animals. Cre/loxP technology was used to generate cell‐specific knockdowns of anoctamin‐1 in ICC (Kit(CreERT2/+);Ano1(tm2jrr/+)) in GI muscles. The recombination efficiency of Kit(CreERT) was evaluated with an eGFP reporter, molecular techniques and immunohistochemistry. Electrical and contractile experiments were used to examine the consequences of anoctamin‐1 knockdown on pacemaker activity, mechanical responses, gastric motility patterns, gastric emptying and GI transit. Reduced anoctamin‐1 caused loss of gastric, but not intestinal slow waves. Irregular spike complexes developed in gastric muscles, leading to uncoordinated antral contractions, delayed gastric emptying and increased total GI transit time. Slow waves in intestinal muscles of juvenile mice were more sensitive to anoctamin‐1 antagonists than slow waves in adult muscles. The low susceptibility to anoctamin‐1 knockdown and weak efficacy of anoctamin‐1 antagonists in inhibiting slow waves in adult small intestinal muscles suggest that a conductance in addition to anoctamin‐1 may develop in small intestinal ICC with ageing and contribute to pacemaker activity.

Smooth muscle layers of the gastrointestinal tract consist of a heterogeneous population of cells that include enteric neurons, several classes of interstitial cells of mesenchymal origin, a variety of immune cells and smooth muscle cells (SMCs). Over the last number of years the complexity of the interactions between these cell types has begun to emerge. For example, interstitial cells, consisting of both interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor alpha-positive (PDGFRα(+)) cells generate pacemaker activity throughout the gastrointestinal (GI) tract and also transduce enteric motor nerve signals and mechanosensitivity to adjacent SMCs. ICC and PDGFRα(+) cells are electrically coupled to SMCs possibly via gap junctions forming a multicellular functional syncytium termed the SIP syncytium. Cells that make up the SIP syncytium are highly specialized containing unique receptors, ion channels and intracellular signaling pathways that regulate the excitability of GI muscles. The unique role of these cells in coordinating GI motility is evident by the altered motility patterns in animal models where interstitial cell networks are disrupted. Although considerable advances have been made in recent years on our understanding of the roles of these cells within the SIP syncytium, the full physiological functions of these cells and the consequences of their disruption in GI muscles have not been clearly defined. This review gives a synopsis of the history of interstitial cell discovery and highlights recent advances in structural, molecular expression and functional roles of these cells in the GI tract.

Kit immunohistochemistry and confocal reconstructions have provided detailed 3-dimensional images of ICC networks throughout the gastrointestinal (GI) tract. Morphological criteria have been used to establish that different classes of ICC exist within the GI tract and physiological studies have shown that these classes have distinct physiological roles in GI motility. Structural studies have focused predominately on rodent models and less information is available on whether similar classes of ICC exist within the GI tracts of humans or non-human primates. Using Kit immunohistochemistry and confocal imaging, we examined the 3-dimensional structure of ICC throughout the GI tract of cynomolgus monkeys. Whole or flat mounts and cryostat sections were used to examine ICC networks in the lower esophageal sphincter (LES), stomach, small intestine and colon. Anti-histamine antibodies were used to distinguish ICC from mast cells in the lamina propria. Kit labeling identified complex networks of ICC populations throughout the non-human primate GI tract that have structural characteristics similar to that described for ICC populations in rodent models. ICC-MY formed anastomosing networks in the myenteric plexus region. ICC-IM were interposed between smooth muscle cells in the stomach and colon and were concentrated within the deep muscular plexus (ICC-DMP) of the intestine. ICC-SEP were found in septal regions of the antrum that separated circular muscle bundles. Spindle-shaped histamine(+) mast cells were found in the lamina propria throughout the GI tract. Since similar sub-populations of ICC exist within the GI tract of primates and rodents and the use of rodents to study the functional roles of different classes of ICC is warranted.

Background Morphological studies have revealed a close anatomical relationship between enteric nerve terminals and intramuscular ICC (ICC-IM) which supports a role for ICC-IM as intermediaries in enteric motor neurotransmission. Recently, a second type of interstitial cell previously described as ‘fibroblast-like’ but can now be identified by platelet-derived growth factor receptor-α expression, has also been implicated in enteric neurotransmission in rodents. The present study was performed to determine if enteric nerve fibers form close anatomical relationships with ICC and PDGFRα+ cells throughout the primate GI tract. Methods Immunohistochemical experiments and confocal microscopy were performed to examine the relationship between excitatory and inhibitory motor neurons, ICC and PDGFRα+ cells throughout the monkey GI tract. Key Results The pan neuronal marker. Protein gene product 9.5 (PGP9.5) was used to label all enteric neurons and substance-P (sub-P) and neuronal nitric oxide synthase (nNOS) to label excitatory and inhibitory neurons, respectively. Double labeling with Kit revealed that both classes of nerve fibers were closely apposed with ICC-IM in the stomach, small intestine and colon (taenia and inter-taenia regions), but not with ICC at the level of the myenteric plexus (ICC-MY). Varicose enteric nerve fibers were closely associated with ICC-IM for distances up to 250 μm. Both excitatory and inhibitory nerve fibers were also closely apposed to PDGFRα+ cells throughout the primate GI tract. Conclusions & Inferences The close anatomical relationship between enteric nerve fibers and ICC-IM and PDGFRα+ cells throughout the GI tract of the Cynomolgus monkey provides morphological evidence that these two classes of interstitial cells may provide a similar physiological function in primates as has been attributed in rodent animal models.

Activation of enteric inhibitory motor neurons causes inhibitory junctional potentials (IJPs) and muscle relaxation in mammalian gastrointestinal (GI) muscles, including humans. IJPs in many GI muscles are bi-phasic with a fast initial hyperpolarization (fIJP) due to release of a purine neurotransmitter and a slower hyperpolarization component (sIJP) due to release of nitric oxide. We sought to characterize the nature of the post-junctional receptor(s) involved in transducing purinergic neural inputs in the murine colon using mice with genetically deactivated P2ry1. Wild-type mice had characteristic biphasic IJPs and pharmacological dissection confirmed that the fIJP was purinergic and the sIJP was nitrergic. The fIJP was completely absent in P2ry1−/− mice and the P2Y1 receptor antagonist MRS2500 had no effect on electrical activity or responses to electrical field stimulation of intrinsic nerves in these mice. Contractile experiments confirmed that purinergic responses were abolished in P2ry1−/− mice. Picospritzing of neurotransmitter candidates (ATP and its primary metabolite, ADP) and β-NAD (and its primary metabolite, ADP-ribose, ADPR) caused transient hyperpolarization responses in wild-type colons, but responses to β-NAD and ADPR were completely abolished in P2ry1−/− mice. Hyperpolarization and relaxation responses to ATP and ADP were retained in colons of P2ry1−/− mice. Video imaging revealed that transit of fecal pellets was significantly delayed in colons from P2ry1−/− mice. These data demonstrate the importance of purinergic neurotransmission in regulating colonic motility and confirm pharmacological experiments suggesting that purinergic neurotransmission is mediated via P2Y1 receptors.

Specialized cells, referred to as interstitial cells of Cajal (ICC), generate and actively propagate the spontaneous electrical activity known as slow waves in the gastrointestinal (GI) tract (Ward et al. 1994, Huizinga et al. 1995; Sanders, 1996). Slow waves time the phasic contractions of GI muscles and provide the underlying organization of excitability for gastric peristalsis and intestinal segmentation. ICC are also interposed between nerve terminals and smooth muscle cells and serve as sites of post-junctional transduction of responses to enteric motor neurotransmitters (see Burns et al. 1996; Ward et al. 2000a). There are at least two classes of ICC, those intermingled within muscle bundles of the circular (CM) and longitudinal muscle (LM) layers (ICC-IM), and those arranged in a dense network between the CM and LM (ICC-MY), at the submucosal surface of the CM (ICC-SM) and in the septa between bundles of smooth muscle cells (ICC-SEP). There have been many studies regarding the mechanisms of pacemaker activity and slow wave propagation, but the precise mechanisms of this behaviour remain controversial. Studies of pacemaker activity in intact muscle strips and bundles have suggested the involvement of a Ca2+-dependent inward current because activity was reduced when the muscles were treated with membrane-permeable Ca2+ buffers (Edwards et al. 1999; Hirst et al. 2002; Kito & Suzuki, 2003). The Ca2+-dependent conductance has been thought to be a Cl− conductance, since a variety of Cl− channel blocking drugs reduced pacemaker activity in guinea-pig and murine muscles (see Hirst et al. 2002; Kito et al. 2002a; Kito & Suzuki, 2003). Similar conclusions were reached from studies of slow waves recorded directly from ICC-MY of the small intestine (e.g. Kito & Suzuki, 2003). In contrast, studies of isolated and cultured ICC have suggested that spontaneous activity is generated by activation of a non-selective cation conductance (Ward et al. 2000; Koh et al. 2002; Sanders et al. 2006), and the putative conductance was found to be inhibited by Ca2+ (Koh et al. 2002). Thus, pacemaker current may be initiated by a transient reduction in [Ca2+]i in a sub-compartment under the plasma membrane containing the non-selective cation conductance (Sanders et al. 2006). No Ca2+-activated inward currents were observed in cultured ICC, and the non-selective cation channels activated by reduced Ca2+ were inhibited by niflumic acid (Koh et al. 2002). Thus, use of Cl− channel antagonists does not necessarily indicate a role for Ca2+- activated Cl− channels in pacemaker activity. A microarray genetic screen recently revealed that Tmem16a is expressed at far greater levels in ICC than in the rest of the muscularis (Chen et al. 2007). Tmem16a encodes ANO1, a Ca2+-activated Cl− channel (Caputo et al. 2008; Schroeder et al. 2008; Yang et al. 2008), and immunohistochemical studies have documented expression of ANO1 (also known as DOG1) protein by ICC (Espinosa et al. 2008; Gomez-Pinilla et al. 2009). Taken together these data suggest the hypothesis that expression and function of these channels may be important in pacemaker activity in the GI tract. Therefore, we have characterized expression of Tmem16a transcripts and ANO1 protein in the tunica muscularis of mouse, monkey (Macaca fascicularis) and human GI tracts using RT-PCR, amplicon sequencing and immunohistochemical techniques. We also evaluated the electrical activity of murine gastric and small intestine muscles, and tested the effects of Cl− channel-blocking drugs. Finally, we tested whether slow wave activity is affected in mice homozygous with null Tmem16a alleles (Tmem16atm1Bdh/tm1Bdh, see Rock et al. 2008). Our data show ubiquitous expression of ANO1 in ICC throughout the GI tract and inhibitory effects of Cl− channel blocking drugs on slow waves. Tmem16a−/− animals failed to generate slow waves, and pacemaker activity did not develop in organ culture after birth, as occurs in wildtype muscles. Together with voltage-clamp studies of isolated ICC (Zhu et al. 2009), our findings strongly support a role for ANO1 in the generation of slow wave currents of GI ICC and electrical slow waves in intact muscles. The model of pacemaker activity deduced from previous studies of cultured ICC (e.g. as detailed in Sanders et al. 2006) will require reconsideration in light of these new findings.

Objective There is a growing body of evidence that gastrointestinal smooth muscle excitability is regulated by several different classes of interstitial cells [interstitial cells of Cajal (ICC) and PDGFRα(+) cells] that are electrically coupled to SMC. Thus, ‘myogenic’ activity results from the integrated behavior of the SMC/ICC/PDGFRα(+) cell (SIP) syncytium. Inputs from excitatory and inhibitory motor neurons are required to produce the complex motor patterns of the gut and coordinate GI motility. Motor neurons innervate these three cell types in the SIP syncytium, and receptors, second messenger pathways, and ion channels in these cells mediate postjunctional responses. Cholinergic neurotransmission in GI muscles from several species has long been thought to be dependent upon activation of a non-selective cation conductance in smooth muscle cells and the molecular candidates for mediating cholinergic excitation have been reported to be the transient receptor protein channels Trpc4 and Trpc6. However, we have shown that cholinergic responses in the GI tract involve ICC and in their absence these motor responses are greatly diminished or absent. We sought to determine the conductance(s) responsible for cholinergic motor responses in the colon. Methods Cre-LoxP recombinase technology was utilized to determine the role of the calcium-activated chloride conductance, Ano1 in post-junctional motor responses in the mouse colon in a cell-specific manner (Kit+ ICC). c-KitCreERT2/+ (Kit-Cre) mice and Ano f/f mice were crossed to generate c-KitCreERT2/+; Ano1f/f (mutants) and c-KitCreERT2/+; Ano1f/+ (controls) animals that were subsequently treated with tamoxifen to induce Cre recombinase expression in ICC. Confocal microscopy was used to determine the cell type Cre expression was switched on. Intracellular microelectrode recordings were performed to determine changes in post-junctional neural responses to nerve stimulation in c-KitCreERT2/+ (Kit-Cre) mice and Anof/f mice to generate c-KitCreERT2/+; Ano1f/f and c-KitCreERT2/+; Ano1f/+ animals mice treated with tamoxifen using electrical field stimulation (EFS). Results Knock down of Ano1 in Kit+ ICC using the Cre/Lox P technology caused a marked reduction or loss of excitatory junction potentials (EJPs) in colonic muscles of c-KitCreERT2/+; Ano1f/f in response to EFS compared to c-KitCreERT2/+; Ano1f/+ controls. These EJPs were atropine sensitive providing evidence that cholinergic muscarinic receptors were responsible for these excitatory responses. Conclusions These data provide evidence that Ano1 expressed in ICC and not smooth muscle cells is critically important for cholinergic excitatory post-junctional neural responses in colonic muscles. Funding Sources NIH PO1 41315 and RO1 DK57236

Serum response factor (SRF) transcriptionally regulates expression of contractile genes in smooth muscle cells (SMC). Lack or decrease of SRF is directly linked to a phenotypic change of SMC, leading to hypomotility of smooth muscle in the gastrointestinal (GI) tract. However, the molecular mechanism behind SRF-induced hypomotility in GI smooth muscle is largely unknown. We describe here how SRF plays a functional role in the regulation of the SMC contractility via myotonic dystrophy protein kinase (DMPK) and L-type calcium channel CACNA1C. GI SMC expressed Dmpk and Cacna1c genes into multiple alternative transcriptional isoforms. Deficiency of SRF in SMC of Srf knockout (KO) mice led to reduction of SRF-dependent DMPK, which down-regulated the expression of CACNA1C. Reduction of CACNA1C in KO SMC not only decreased intracellular Ca2+ spikes but also disrupted their coupling between cells resulting in decreased contractility. The role of SRF in the regulation of SMC phenotype and function provides new insight into how SMC lose their contractility leading to hypomotility in pathophysiological conditions within the GI tract.

Background & Aims Interstitial cells of Cajal (ICC) express the receptor tyrosine kinase, KIT, the receptor for stem cell factor. In the gastrointestinal (GI) tract, ICC are pacemaker cells that generate spontaneous electrical slow waves, and mediate inputs from motor neurons. Absence or loss of ICC are associated with GI motility disorders, including those consequent of diabetes. Studies of ICC have been hampered by the low density of these cells and difficulties in recognizing these cells in cell dispersions. Methods Kit +/copGFP mice harboring a copepod super green fluorescent protein ( copGFP ) complementary DNA, inserted at the Kit locus, were generated. copGFP + ICC from GI muscles were analyzed using confocal microscopy and flow cytometry. copGFP + ICC from the jejunum were purified by a fluorescence-activated cell sorter and validated by cell-specific markers. Kit +/copGFP mice were crossbred with diabetic Lep +/ob mice to generate compound Kit +/copGFP ;Lep ob/ob mutant mice. copGFP + ICC from compound transgenic mice were analyzed by confocal microscopy. Results copGFP in Kit +/copGFP mice colocalized with KIT immunofluorescence and thus was predominantly found in ICC. In other smooth muscles, mast cells were also labeled, but these cells were relatively rare in the murine GI tract. copGFP + cells from jejunal muscles were Kit + and free of contaminating cell-specific markers. Kit +/copGFP ; Lep ob/ob mice displayed ICC networks that were dramatically disrupted during the development of diabetes. Conclusions Kit +/copGFP mice offer a powerful new model to study the function and genetic regulation of ICC phenotypes. Isolation of ICC from animal models will help determine the causes and responses of ICC to therapeutic agents.

Phosphoinositide 3-kinase (PI 3-kinase) is a ubiquitous enzyme that catalyses the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-trisphosphate (PIP3). PI 3-kinase driven generation of PIP3 activates various downstream signalling cascades which in turn regulate numerous biological processes including growth, metabolism, proliferation, migration and cell size. PTEN (phosphatase and tensin homologue deleted on chromosome 10), which was originally identified as a tumour promoter, is a constitutively active lipid phosphatase that opposes PI 3-kinase-dependent signalling by promoting dephosphorylation of PIP3 into PIP2. PTEN is highly expressed in neurons and several lines of evidence support a role for PTEN in regulating important neuronal functions. Indeed, familial mutations that result in PTEN inactivation have been linked to neurological disorders such as ataxia, mental retardation and seizures (Backman et al. 2001). Moreover, loss of PTEN function at early stages of development results in widespread deficits in neuronal growth, synaptogenesis and synaptic plasticity suggesting additional roles for PTEN in these processes. However, it is not clear if PTEN-linked synaptic impairments are a direct consequence of loss of PTEN function or are secondary to PTEN-driven alterations in neuronal morphology. A recent insightful study by Sperow et al. (2012) in The Journal of Physiology aims to address this issue. This study demonstrates that the structural and functional properties of hippocampal synapses are independently controlled by PTEN and that PTEN plays a direct role in activity-dependent hippocampal synaptic plasticity. Several studies have observed that a key feature of deleting PTEN during CNS development is progressive hypertrophy of neuronal dendrites and spines as well as a marked increase in the size of neurons and synapses. PTEN deletion or down-regulation in hippocampal neurons also results in impairments in two forms of activity-dependent synaptic plasticity, namely long-term potentiation (LTP) and long-term depression (LTD). In order to determine if the structural and functional effects of PTEN on hippocampal synapses are related, Sperow and colleagues generated transgenic mice with conditional knockout of PTEN in forebrain excitatory neurons. As PTEN deletion was apparent after postnatal day 14 (P14) in these mice, the impact of PTEN deletion on hippocampal synaptic function could be examined prior to its hypertrophic effects. Postnatal deletion of PTEN had no significant effect on excitatory synaptic transmission at hippocampal CA1 synapses, as basal synaptic transmission and paired pulse facilitation was normal in PTEN−/− mice compared to wild-type littermates. In contrast, deficits in both hippocampal LTP and LTD were observed in PTEN−/− mice. Postnatal deletion of PTEN also resulted in hippocampal-specific memory deficits as significant impairments in spatial memory tasks performed in the Morris water maze were observed in PTEN−/− mice. However, no differences in soma size or dendritic morphology were detected between PTEN+ and PTEN− neurons in PTEN−/− mice, suggesting that structural abnormalities do not contribute to the deficits in synaptic plasticity following postnatal PTEN deletion. As PTEN negatively regulates PI 3-kinase driven signalling, deletion or inhibition of PTEN results in uncontrolled stimulation of downstream components of the PI 3-kinase cascade, including the protein kinase PDK1. Consequently reducing PDK1 expression can reverse some neuronal abnormalities linked to PTEN deletion (Chalhoub et al. 2009). In this study, Sperow et al. examined whether postnatal deletion of PDK1 rescued the impairments in hippocampal synaptic plasticity in PTEN−/− mice. Postnatal deletion of PDK1 had no effect on hippocampal synaptic plasticity or neuronal morphology per se. However, deletion of PDK1 rescued the synaptic plasticity deficits, as the magnitude of hippocampal LTP and LTD was comparable in double mutant mice (PTEN−/−; PDK1−/−) and wild-type littermates. In contrast, however, significant impairments in spatial memory were still observed in the double mutant mice compared to wild-type littermates, suggesting that PDK1 deletion is able to counterbalance the effects of postnatal PTEN deletion on synaptic plasticity but not on spatial memory. In conclusion this study indicates that hippocampal synaptic plasticity and neuronal morphology are independently regulated by PTEN in vivo. It also highlights an important role for PTEN in hippocampal LTP and LTD. Although the precise cellular mechanisms underlying PTEN modulation of synaptic plasticity are not fully known, recent studies suggest involvement of a postsynaptic mechanism as PTEN inhibition promotes AMPA receptor trafficking to synapses leading to a persistent increase in excitatory synaptic strength in adult hippocampal slices (Moult et al. 2010). Conversely enhanced PTEN lipid phosphatase activity has been reported to depress excitatory synaptic transmission, which is in turn required for NMDA receptor-dependent LTD (Jurado et al. 2011). Together these findings provide important new insights into the role of PTEN in regulating hippocampal synaptic function.