The Role of AMPARs in the Maturation and Integration of Caudal Ganglionic Eminence-Derived Interneurons into Developing Hippocampal Microcircuits.

In the hippocampal CA1, caudal ganglionic eminence (CGE)-derived interneurons are recruited by activation of glutamatergic synapses comprising GluA2-containing calcium-impermeable AMPARs and exert inhibitory regulation of the local microcircuit. However, the role played by AMPARs in maturation of the developing circuit is unknown. We demonstrate that elimination of the GluA2 subunit (GluA2 KO) of AMPARs in CGE-derived interneurons, reduces spontaneous EPSC frequency coupled to a reduction in dendritic glutamatergic synapse density. Removal of GluA1&2&3 subunits (GluA1-3 KO) in CGE-derived interneurons, almost completely eliminated sEPSCs without further reducing synapse density, but increased dendritic branching. Moreover, in GluA1-3 KOs, the number of interneurons invading the hippocampus increased in the early postnatal period but converged with WT numbers later due to increased apoptosis. However, the CCK-containing subgroup increased in number, whereas the VIP-containing subgroup decreased. Both feedforward and feedback inhibitory input onto pyramidal neurons was decreased in GluA1-3 KO. These combined anatomical, synaptic and circuit alterations, were accompanied with a wide range of behavioural abnormalities in GluA1-3 KO mice compared to GluA2 KO and WT. Thus, AMPAR subunits differentially contribute to numerous aspects of the development and maturation of CGE-derived interneurons and hippocampal circuitry that are essential for normal behaviour.

Afferent specific role of NMDA receptors for the circuit integration of hippocampal neurogliaform cells.

Appropriate integration of GABAergic interneurons into nascent cortical circuits is critical for ensuring normal information processing within the brain. Network and cognitive deficits associated with neurological disorders, such as schizophrenia, that result from NMDA receptor-hypofunction have been mainly attributed to dysfunction of parvalbumin-expressing interneurons that paradoxically express low levels of synaptic NMDA receptors. Here, we reveal that throughout postnatal development, thalamic, and entorhinal cortical inputs onto hippocampal neurogliaform cells are characterized by a large NMDA receptor-mediated component. This NMDA receptor-signaling is prerequisite for developmental programs ultimately responsible for the appropriate long-range AMPAR-mediated recruitment of neurogliaform cells. In contrast, AMPAR-mediated input at local Schaffer-collateral synapses on neurogliaform cells remains normal following NMDA receptor-ablation. These afferent specific deficits potentially impact neurogliaform cell mediated inhibition within the hippocampus and our findings reveal circuit loci implicating this relatively understudied interneuron subtype in the etiology of neurodevelopmental disorders characterized by NMDA receptor-hypofunction.Proper brain function depends on the correct assembly of excitatory and inhibitory neurons into neural circuits. Here the authors show that during early postnatal development in mice, NMDAR signaling via activity of long-range synaptic inputs onto neurogliaform cells is required for their appropriate integration into the hippocampal circuitry.

Persistent inhibitory circuit defects and disrupted social behaviour following in utero exogenous cannabinoid exposure.

Placental transfer of Δ9-tetrahydrocannabinol (THC) during pregnancy has the potential to interfere with endogenous cannabinoid (CB) regulation of fetal nervous system development in utero. Here we examined the effect of maternal CB intake on mouse hippocampal interneurons largely focusing on cholecystokinin-expressing interneurons (CCK-INTs), a prominent CB subtype-1 receptor (CB1R) expressing neuronal population throughout development. Maternal treatment with THC or the synthetic CB1R agonist WIN55,212-2 (WIN) produced a significant loss of CCK-INTs in the offspring. Further, residual CCK-INTs in animals prenatally treated with WIN displayed decreased dendritic complexity. Consistent with these anatomical deficits, pups born to CB-treated dams exhibited compromised CCK-INT-mediated feedforward and feedback inhibition. Moreover, pups exposed to WIN in utero lacked constitutive CB1R-mediated suppression of inhibition from residual CCK-INTs and displayed altered social behavior. Our findings add to a growing list of potential cell/circuit underpinnings that may underlie cognitive impairments in offspring of mothers that abuse marijuana during pregnancy.

FISHing Tips: What Every Clinician Should Know About 1p19q Analysis in Gliomas Using Fluorescence in situ Hybridisation.

1p19q co-deletion is a chromosomal alteration associated with primary brain tumours of oligodendroglial histology. It is an established predictive and prognostic biomarker that informs whether patients are offered radiotherapy, chemotherapy or both. In the near future, 1p19q co-deletion status may also be incorporated into the reclassification of gliomas. Analysis is commonly carried out using fluorescence in situ hybridisation (FISH) because it is a reliable and validated laboratory technique. The result is generally considered to be dichotomous (1p19q co-deletion present or absent), but there are subtleties in interpretation that are of clinical relevance. Separate centres may interpret certain chromosome deletion patterns differently. Pivotal trials in mixed and pure anaplastic oligodendrogliomas have used slightly different FISH probe ratios as the cut-off for chromosome deletion. Here we review the clinical implications of this variability and review the process of 1p19q co-deletion assessment using FISH in gliomas from a clinician's perspective. We also consider common alternative methods of analysis.

The concepts, diagnosis and management of early imaging changes after therapy for glioblastomas.

Since postoperative radiotherapy plus concomitant temozolomide followed by adjuvant temozolomide has become standard treatment for glioblastoma, the phenomenon of early post-treatment enlargement of the imaged tumour volume, usually without clinical deterioration, has become widely recognised. The term pseudoprogression has been used to describe a poorly understood pathophysiological process. In this review, the pathophysiological concepts, relevance, diagnosis and management of patients with 'pseudoprogression' and 'pseudoresponse' are discussed. Guidelines are given with respect to radiological imaging modality, mode and frequency. Further biological and clinical insights into these phenomena require carefully designed prospective studies.

Burst firing induces postsynaptic LTD at developing mossy fibre-CA3 pyramid synapses.

Synaptic development is an activity-dependent process utilizing coordinated network activity to drive synaptogenesis and subsequent refinement of immature connections. Hippocampal CA3 pyramidal neurons (PYRs) exhibit intense burst firing (BF) early in development, concomitant with the period of mossy fibre (MF) development. However, whether developing MF-PYR synapses utilize PYR BF to promote MF synapse maturation remains unknown. Recently, we demonstrated that transient tonic depolarization of postsynaptic PYRs induces a persistent postsynaptic form of long-term depression (depolarization-induced long-term depression, DiLTD) at immature MF-PYR synapses. DiLTD induction is NMDAR independent but does require postsynaptic Ca(2+) influx through L-type voltage gated Ca(2+) channels (L-VGCCs), and is expressed as a reduction in AMPAR function through the loss of GluR2-lacking AMPARs present at immature MF-PYR synapses. Here we examined whether more physiologically relevant phasic L-VGCC activation by PYR action potential (AP) BF activity patterns can trigger DiLTD. Using combined electrophysiological and Ca(2+) imaging approaches we demonstrate that PYR BF effectively drives L-VGCC activation and that brief periods of repetitive PYR BF, produced by direct current injection or intrinsic network activity induces NMDAR-independent LTD by promoting Ca(2+) influx through the activated L-VGCCs. This BF induced LTD, just like DiLTD, is specific for developing MF-PYR synapses, is PICK1 dependent, and is expressed postsynaptically. Our results demonstrate that DiLTD can be induced by phasic L-VGCC activation driven by PYR BF, suggesting the engagement of natural PYR network activity patterns for MF synapse maturation.

Ultrasound imaging to assess inter- and intra-fraction motion during bladder radiotherapy and its potential as a verification tool.

AIMS: Organ motion is the principle source of error in bladder cancer radiotherapy. The aim of this study was to evaluate ultrasound bladder volume measurement as a surrogate measure of organ motion during radiotherapy: (1) to assess inter- and intra-fraction bladder variation and (2) as a potential treatment verification tool. MATERIALS AND METHODS: Twenty patients receiving radical radiotherapy for bladder cancer underwent post-void ultrasound bladder volume measurement at the time of radiotherapy treatment planning (RTP), and immediately before (post-void) and after receiving daily fractions. RESULTS: Ultrasound bladder volume measurement was found to be a simple and acceptable method to estimate relative bladder volume changes. Six patients showed significant changes to post-void bladder volume over the treatment course (P<0.05). The mean inter-fraction post-void bladder volume of five patients exceeded their RTP ultrasound bladder volume by more than 50%. Intra-fraction bladder volume increased on 275/308 (89%) assessed fractions, with the mean intra-fraction volume increases of seven patients exceeding their RTP ultrasound bladder volume by more than 50%. CONCLUSIONS: Both day-to-day bladder volume variation and bladder filling during treatment should be considered in RTP and delivery. Ultrasound may provide a practical daily verification tool by: supporting volume limitation as a method of treatment margin reduction; allowing detection of patients who may require interventions to promote bladder reproducibility; and identifying patients with prominent volume changes for the selective application of more advanced adaptive/image-guided radiotherapy techniques.

Early clinical evaluation of a novel three-dimensional structure delineation software tool (SCULPTER) for radiotherapy treatment planning.

Modern radiotherapy treatment planning (RTP) necessitates increased delineation of target volumes and organs at risk. Conventional manual delineation is a laborious, time-consuming and subjective process. It is prone to inconsistency and variability, but has the potential to be improved using automated segmentation algorithms. We carried out a pilot clinical evaluation of SCULPTER (Structure Creation Using Limited Point Topology Evidence in Radiotherapy) - a novel prototype software tool designed to improve structure delineation for RTP. Anonymized MR and CT image datasets from patients who underwent radiotherapy for bladder or prostate cancer were studied. An experienced radiation oncologist used manual and SCULPTER-assisted methods to create clinically acceptable organ delineations. SCULPTER was also tested by four other RTP professionals. Resulting contours were compared by qualitative inspection and quantitatively by using the volumes of the structures delineated and the time taken for completion. The SCULPTER tool was easy to apply to both MR and CT images and diverse anatomical sites. SCULPTER delineations closely reproduced manual contours with no significant volume differences detected, but SCULPTER delineations were significantly quicker (p<0.05) in most cases. In conclusion, clinical application of SCULPTER resulted in rapid and simple organ delineations with equivalent accuracy to manual methods, demonstrating proof-of-principle of the SCULPTER system and supporting its potential utility in RTP.

Radiation therapy for muscle-invasive bladder cancer: treatment planning and delivery.

Radiation therapy allows organ preservation therapy in selected patients with muscle invasive bladder cancer. This review sets out to explore the role of radiation therapy in the management of muscle invasive bladder cancer. It describes and assesses the potential benefits provided by technological advances in radiation delivery in optimizing the therapeutic ratio.

Hypofractionated radiotherapy as salvage for rising prostate-specific antigen after radical prostatectomy.

AIMS: To review the outcome of men receiving hypofractionated salvage radiotherapy for rising prostate-specific antigen (PSA) after radical prostatectomy. MATERIALS AND METHODS: A retrospective analysis of 61 men referred for salvage radiotherapy for biochemical relapse after radical prostatectomy was conducted. Twenty-four men receiving hormonal therapy or with follow-up of less than 12 months were excluded. Thirty-seven men were identified, median age 64 years, median preoperative PSA 11 ng/ml (5.6-60 ng/ml), Gleason scores <7: 70%, Gleason scores > or = 7: 30%. Twenty-seven men had positive surgical resection margins, eight had seminal-vesicle involvement and one had lymph-node involvement. Diagnosis of failure after radical prostatectomy was made on rising PSA in all cases; 19 men also had positive magnetic resonance imaging, 11 abnormal digital rectal examination and nine positive biopsy. Radiotherapy was delivered conformally to the prostatic fossa, 50-52.5 Gy in 20 fractions over 4 weeks. Date of failure after radiotherapy was defined by the American Society for Therapeutic Radiology and Oncology (ASTRO) consensus criteria or as date of commencement of hormonal therapy for rising PSA. RESULTS: Median time from radical prostatectomy to radiotherapy was 30.6 months (8-68 months); median pre-radiotherapy PSA was 2.9 ng/ml (0.5-11.4 ng/ml). PSA response after radiotherapy was seen in 33 out of 37 (89%) patients. At median follow-up of 36 months (20-85 months), 28 out of 37 remained disease-free. Thirteen more patients have had two consecutive PSA rises. Actuarial 3-year disease-free survival is 74%. No patient has developed metastases or died of prostate cancer. Pre-radiotherapy PSA less than 2 ng/ml predicted disease-free survival (P = 0.027). No acute toxicity greater than Radiation Therapy Oncology Group (RTOG) G2 was observed. CONCLUSIONS: Salvage radiotherapy after radical prostatectomy achieved durable biochemical control in most patients. Outcome is improved if radiotherapy is delivered when PSA is less than 2 ng/ml. A policy of close monitoring after radical prostatectomy with early referral for salvage radiotherapy is advocated.

Local recurrence of breast cancer following surgery and radiotherapy: incidence and outcome.

Local recurrence of cancer in the treated breast following breast-conserving surgery and radiotherapy occurs in a minority of patients, but can represent a significant clinical problem. The impact of local relapse on the subsequent course of the disease is disputed. The aim of this retrospective review was to identify the rate and prognostic factors for breast recurrence and to determine the subsequent outcome. The case notes of 2159 patients treated between 1989 and 1992 were reviewed. Actuarial local relapse rate was 6.3% at 5 years. Factors predictive for recurrence on multivariate analysis were age (P<0.001), status of excision margins (P=0.019), and pathological UICC stage (P=0.01). One hundred and sixty-one patients developed local recurrence in the treated breast of whom 101 were treated with further surgery. The 5-year cancer-specific survival of this group was comparable with that of the patients who remained free of breast relapse (82 vs. 88%) but subsequently fell to 61 vs. 80% at 8 years (P<0.001). Sixty patients were unable to have salvage surgery; their cancer-specific survival was much worse than that of patients with operable recurrences at 33% at 5 years and 13% at 8 years. Eighty-three patients (4% of the original 2159 patients) had uncontrolled local disease at time of death or last follow-up. The prognosis of patients who developed recurrence within 2 years of their initial treatment was inferior to those who developed recurrences after 4 years (cancer-specific survival 5 years post-recurrence 23 vs. 57% P=0.008). Systemic therapy should be considered for patients with early breast recurrence in view of their inferior survival.

Regulation of Kv1 subunit expression in oligodendrocyte progenitor cells and their role in G1/S phase progression of the cell cycle.

Proliferative oligodendrocyte progenitor cells (OPs) express large, delayed outward-rectifying K(+) currents (I(K)), whereas nondividing immature and mature oligodendrocytes display much smaller I(K). Here, we show that up-regulation of I(K) occurs in G(1) phase of the cell cycle in purified cultured OPs and is the result of an RNA synthesis-dependent, selective increase of the K(+) channel subunit proteins Kv1.3 and Kv1.5. In oligodendrocyte cells acutely isolated from developing rat brain, a decrease of cyclin D expression is observed as these cells mature along their lineage. This is accompanied by a decrease in Kv1.3 and Kv1.5 subunit expression, suggesting a role for these subunits in the proliferative potential of OPs in situ. I(K) expressed in OPs in subventricular zone and developing white matter in acutely isolated slice preparations were selectively blocked by antagonists of Kv1.3, illustrating the functional presence of this subunit in situ. Interestingly, Kv1.3 block inhibited S-phase entry of both purified OPs in culture and in tissue slice cultures. Thus, we employ both in vitro and in situ experimental approaches to show that (i) RNA-dependent synthesis of Kv1.3 and Kv1.5 subunit proteins occurs in G(1) phase of the OP cell cycle and is responsible for the observed increase in I(K), and (ii) currents through Kv1.3-containing channels play a crucial role in G(1)/S transition of proliferating OPs.

Differential synaptic processing separates stationary from transient inputs to the auditory cortex.

Sound features are blended together en route to the central nervous system before being discriminated for further processing by the cortical synaptic network. The mechanisms underlying this synaptic processing, however, are largely unexplored. Intracortical processing of the auditory signal was investigated by simultaneously recording from pairs of connected principal neurons in layer II/III in slices from A1 auditory cortex. Physiological patterns of stimulation in the presynaptic cell revealed two populations of postsynaptic events that differed in mean amplitude, failure rate, kinetics and short-term plasticity. In contrast, transmission between layer II/III pyramidal neurons in barrel cortex were uniformly of large amplitude and high success (release) probability (Pr). These unique features of auditory cortical transmission may provide two distinct mechanisms for discerning and separating transient from stationary features of the auditory signal at an early stage of cortical processing.

Kv3 channels: voltage-gated K+ channels designed for high-frequency repetitive firing.

Analysis of the Kv3 subfamily of K(+) channel subunits has lead to the discovery of a new class of neuronal voltage-gated K(+) channels characterized by positively shifted voltage dependencies and very fast deactivation rates. These properties are adaptations that allow these channels to produce currents that can specifically enable fast repolarization of action potentials without compromising spike initiation or height. The short spike duration and the rapid deactivation of the Kv3 currents after spike repolarization maximize the quick recovery of resting conditions after an action potential. Several neurons in the mammalian CNS have incorporated into their repertoire of voltage-dependent conductances a relatively large number of Kv3 channels to enable repetitive firing at high frequencies - an ability that crucially depends on the special properties of Kv3 channels and their impact on excitability.

Interneurons unbound.

Local-circuit, gamma-aminobutyric acid-releasing inhibitory interneurons of the hippocampus and cortex have traditionally been considered as the regulators of principal neuron activity--the yin to the excitatory yang. Recent evidence indicates that, in addition to that role, their network connectivity and the properties of their intrinsic voltage-gated currents are finely tuned to permit inhibitory interneurons to generate and control the rhythmic output of large populations of both principal cells and other populations of inhibitory interneurons. This review brings together recently described properties and emerging principles of interneuron function that indicate a much more complex role for these cells than just providers of inhibition.

Differential mechanisms of transmission at three types of mossy fiber synapse.

The axons of the dentate gyrus granule cells, the so-called mossy fibers, innervate their inhibitory interneuron and pyramidal neuron targets via both anatomically and functionally specialized synapses. Mossy fiber synapses onto inhibitory interneurons were comprised of either calcium-permeable (CP) or calcium-impermeable (CI) AMPA receptors, whereas only calcium-impermeable AMPA receptors existed at CA3 principal neuron synapses. In response to brief trains of high-frequency stimuli (20 Hz), pyramidal neuron synapses invariably demonstrated short-term facilitation, whereas interneuron EPSCs demonstrated either short-term facilitation or depression. Facilitation at all CI AMPA synapses was voltage independent, whereas EPSCs at CP AMPA synapses showed greater facilitation at -20 than at -80 mV, consistent with a role for the postsynaptic unblock of polyamines. At pyramidal cell synapses, mossy fiber EPSCs possessed marked frequency-dependent facilitation (commencing at stimulation frequencies >0.1 Hz), whereas EPSCs at either type of interneuron synapse showed only moderate frequency-dependent facilitation or underwent depression. Presynaptic metabotropic glutamate receptors (mGluRs) decreased transmission at all three synapse types in a frequency-dependent manner. However, after block of presynaptic mGluRs, transmission at interneuron synapses still did not match the dynamic range of EPSCs at pyramidal neuron synapses. High-frequency stimulation of mossy fibers induced long-term potentiation (LTP), long-term depression (LTD), or no change at pyramidal neuron synapses, interneuron CP AMPA synapses, and CI AMPA synapses, respectively. Induction of LTP or LTD altered the short-term plasticity of transmission onto both pyramidal cells and interneuron CP AMPA synapses by a mechanism consistent with changes in release probability. These data reveal differential mechanisms of transmission at three classes of mossy fiber synapse made onto distinct targets.

Snap-25 is polarized to axons and abundant along the axolemma: an immunogold study of intact neurons.

SNAP-25, synaptosomal associated protein of 25 kDa, is reported to be a t-SNARE (target receptor associated with the presynaptic plasma membrane) involved in the docking and fusion of synaptic vesicles. We present here the first ultrastructural localization of SNAP-25 in intact neurons by pre-embedding EM immunocytochemistry in rat brains, hippocampal slice cultures, and PC12 cells. In differentiated neurons, SNAP-25 labeling was clearly membrane-associated. The labeling was most prominent in the plasma membrane of axons and excluded from the plasma membranes of soma and dendrites. Furthermore, SNAP-25 did not appear to be restricted to the synaptic junctions. SNAP-25 labeling was seen in the cytoplasm of the soma and large dendrites, mostly associated with the Golgi complexes. There were also some SNAP-25 labeled tubulo-vesicular structures in the cytoplasm of the soma and the axons, but rarely in the smaller dendrites. In PC12 cells, after 5-10 minutes of high potassium (75 mM) stimulation in the presence of HRP, SNAP-25 labeling appeared, additionally, on HRP-filled early endosomes. After a longer (20-30 minutes) HRP incubation, most of the later stage endosomes and lysosomes were loaded with HRP but they were negative for SNAP-25. These results suggest that SNAP-25 is sorted out of these late endosomal compartments, and that the bulk of the SNAP-25 protein is probably recycled back to the axolemma from the early endosomes. In contrast, in those samples which were incubated with HRP for longer periods, there were still some SNAP-25-positive vesicular structures which were HRP-negative. These structures most likely represent anterograde vesicles that carry newly synthesized SNAP-25 from the soma to the axolemma by axonal transport. SNAP-25 appears to be sorted at the Golgi complex to reach the axolemma specifically. Its widespread distribution all along the axolemma does not support the view of SNAP-25 as a t-SNARE limited for synaptic exocytosis.

H2 histamine receptor-phosphorylation of Kv3.2 modulates interneuron fast spiking.

Histamine-containing neurons of the tuberomammilary nucleus project to the hippocampal formation to innervate H1 and H2 receptors on both principal and inhibitory interneurons. Here we show that H2 receptor activation negatively modulates outward currents through Kv3.2-containing potassium channels by a mechanism involving PKA phosphorylation in inhibitory interneurons. PKA phosphorylation of Kv3.2 lowered the maximum firing frequency of inhibitory neurons, which in turn negatively modulated high-frequency population oscillations recorded in principal cell layers. All these effects were absent in a Kv3.2 knockout mouse. These data reveal a novel pathway for histamine-dependent regulation of high-frequency oscillations within the hippocampal formation.

Target-specific expression of pre- and postsynaptic mechanisms.

Target-specific expression of pre- and postsynaptic mechanisms of synaptic transmission has been shown in a variety of central neurons by a number of laboratories. These data have demonstrated that synaptic transmission between single axons diverging onto distinct target neurons can behave independently, differentially influencing activity in the target neuron. Similarly, single neurons are capable of manufacturing molecularly distinct ligand-gated receptors and targeting them to synapses innervated by distinct converging afferent projections. A picture is emerging consistent with a role for both pre- and postsynaptic mechanisms in influencing the target-specific nature of transmission at numerous diverse synapses throughout the mammalian CNS. This target specificity adds another level of complexity in unravelling the roles played by individual neurons within a computational network. To begin to understand the coordinated activity of large ensembles of neurons it is becoming clear that the nature of transmission between individual pre- and postsynaptic elements within a circuit must first be understood for each and every neural element involved.