Quantification of Aging-Related Decreases in Sensory Innervation of the Bladder Trigone in Rats.

PURPOSE: The prevalence of lower urinary tract symptoms (LUTS), characterized by problems regarding storage and/or voiding of urine, is known to significantly increase with age. Effective communication between the lower urinary tract and the central nervous system (CNS) is essential for the optimal function of this system, and heavily relies on the efficient interaction between the bladder urothelium and the afferent nerve fibers situated in close proximity to the urothelium within the lamina propria. METHODS: We aimed to quantify aging-related differences in the expression of calcitonin gene-related peptide (CGRP, an established marker for sensory nerve fibers) in the trigonal mucosal layers of young (3-4 months) and aged (25-30 months) rats. We evaluated trigonal tissue from 3 animals per age group. Tissue was serially sectioned at 10 µm and stained for CGRP. Images were taken along the full length of the tissue. For each image we computed the total CGRP-positive area (µm2) and the median value for each animal was used for further analysis. RESULTS: Upon statistical analysis the aged rats show a significantly lower CGRP-positive area compared to young rats (P=0.0049). These results indicate that aging has a negative effect on the area of CGRP-positive signal in the trigone. CONCLUSION: The structural and functional integrity of the sensory web in the trigonum of rats is negatively affected by the aging process, potentially leading to impaired communication between the bladder urothelium the CNS. Consequently, these perturbations in the sensory system may contribute to the pathogenesis or exacerbation LUTS.

Region-specific Foxp2 deletions in cortex, striatum or cerebellum cannot explain vocalization deficits observed in spontaneous global knockouts.

FOXP2 has been identified as a gene related to speech in humans, based on rare mutations that yield significant impairments in speech at the level of both motor performance and language comprehension. Disruptions of the murine orthologue Foxp2 in mouse pups have been shown to interfere with production of ultrasonic vocalizations (USVs). However, it remains unclear which structures are responsible for these deficits. Here, we show that conditional knockout mice with selective Foxp2 deletions targeting the cerebral cortex, striatum or cerebellum, three key sites of motor control with robust neural gene expression, do not recapture the profile of pup USV deficits observed in mice with global disruptions of this gene. Moreover, we observed that global Foxp2 knockout pups show substantive reductions in USV production as well as an overproduction of short broadband noise "clicks", which was not present in the brain region-specific knockouts. These data indicate that deficits of Foxp2 expression in the cortex, striatum or cerebellum cannot solely explain the disrupted vocalization behaviours in global Foxp2 knockouts. Our findings raise the possibility that the impact of Foxp2 disruption on USV is mediated at least in part by effects of this gene on the anatomical prerequisites for vocalizing.

AMPAR Auxiliary Protein SHISA6 Facilitates Purkinje Cell Synaptic Excitability and Procedural Memory Formation.

The majority of excitatory postsynaptic currents in the brain are gated through AMPA-type glutamate receptors, the kinetics and trafficking of which can be modulated by auxiliary proteins. It remains to be elucidated whether and how auxiliary proteins can modulate synaptic function to contribute to procedural memory formation. In this study, we report that the AMPA-type glutamate receptor (AMPAR) auxiliary protein SHISA6 (CKAMP52) is expressed in cerebellar Purkinje cells, where it co-localizes with GluA2-containing AMPARs. The absence of SHISA6 in Purkinje cells results in severe impairments in the adaptation of the vestibulo-ocular reflex and eyeblink conditioning. The physiological abnormalities include decreased presence of AMPARs in synaptosomes, impaired excitatory transmission, increased deactivation of AMPA receptors, and reduced induction of long-term potentiation at Purkinje cell synapses. Our data indicate that Purkinje cells require SHISA6-dependent modification of AMPAR function in order to facilitate cerebellar, procedural memory formation.

Differential effects of Foxp2 disruption in distinct motor circuits.

Disruptions of the FOXP2 gene cause a speech and language disorder involving difficulties in sequencing orofacial movements. FOXP2 is expressed in cortico-striatal and cortico-cerebellar circuits important for fine motor skills, and affected individuals show abnormalities in these brain regions. We selectively disrupted Foxp2 in the cerebellar Purkinje cells, striatum or cortex of mice and assessed the effects on skilled motor behaviour using an operant lever-pressing task. Foxp2 loss in each region impacted behaviour differently, with striatal and Purkinje cell disruptions affecting the variability and the speed of lever-press sequences, respectively. Mice lacking Foxp2 in Purkinje cells showed a prominent phenotype involving slowed lever pressing as well as deficits in skilled locomotion. In vivo recordings from Purkinje cells uncovered an increased simple spike firing rate and decreased modulation of firing during limb movements. This was caused by increased intrinsic excitability rather than changes in excitatory or inhibitory inputs. Our findings show that Foxp2 can modulate different aspects of motor behaviour in distinct brain regions, and uncover an unknown role for Foxp2 in the modulation of Purkinje cell activity that severely impacts skilled movements.

Impact of parallel fiber to Purkinje cell long-term depression is unmasked in absence of inhibitory input.

Pavlovian eyeblink conditioning has been used extensively to study the neural mechanisms underlying associative and motor learning. During this simple learning task, memory formation takes place at Purkinje cells in defined areas of the cerebellar cortex, which acquire a strong temporary suppression of their activity during conditioning. Yet, it is unknown which neuronal plasticity mechanisms mediate this suppression. Two potential mechanisms include long-term depression of parallel fiber to Purkinje cell synapses and feed-forward inhibition by molecular layer interneurons. We show, using a triple transgenic approach, that only concurrent disruption of both these suppression mechanisms can severely impair conditioning, highlighting that both processes can compensate for each other's deficits.

Chloride Homeostasis in Neurons With Special Emphasis on the Olivocerebellar System: Differential Roles for Transporters and Channels.

The intraneuronal ionic composition is an important determinant of brain functioning. There is growing evidence that aberrant homeostasis of the intracellular concentration of Cl- ([Cl-]i) evokes, in addition to that of Na+ and Ca2+, robust impairments of neuronal excitability and neurotransmission and thereby neurological conditions. More specifically, understanding the mechanisms underlying regulation of [Cl-]i is crucial for deciphering the variability in GABAergic and glycinergic signaling of neurons, in both health and disease. The homeostatic level of [Cl-]i is determined by various regulatory mechanisms, including those mediated by plasma membrane Cl- channels and transporters. This review focuses on the latest advances in identification, regulation and characterization of Cl- channels and transporters that modulate neuronal excitability and cell volume. By putting special emphasis on neurons of the olivocerebellar system, we establish that Cl- channels and transporters play an indispensable role in determining their [Cl-]i and thereby their function in sensorimotor coordination.

Impact of NMDA Receptor Overexpression on Cerebellar Purkinje Cell Activity and Motor Learning.

In many brain regions involved in learning NMDA receptors (NMDARs) act as coincidence detectors of pre- and postsynaptic activity, mediating Hebbian plasticity. Intriguingly, the parallel fiber (PF) to Purkinje cell (PC) input in the cerebellar cortex, which is critical for procedural learning, shows virtually no postsynaptic NMDARs. Why is this? Here, we address this question by generating and testing independent transgenic lines that overexpress NMDAR containing the type 2B subunit (NR2B) specifically in PCs. PCs of the mice that show larger NMDA-mediated currents than controls at their PF input suffer from a blockage of long-term potentiation (LTP) at their PF-PC synapses, while long-term depression (LTD) and baseline transmission are unaffected. Moreover, introducing NMDA-mediated currents affects cerebellar learning in that phase-reversal of the vestibulo-ocular reflex (VOR) is impaired. Our results suggest that under physiological circumstances PC spines lack NMDARs postsynaptically at their PF input so as to allow LTP to contribute to motor learning.

Cerebellar and Striatal Pathologies in Mouse Models of Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition with a strong genetic component. To date, several hundred different genetic mutations have been identified to play a role in its aetiology. The heterogeneity of genetic abnormalities combined with the different brain regions where aberrations are found makes the search for causative mechanisms a daunting task. Even within a limited number of brain regions, a myriad of different neural circuit dysfunctions may lead to ASD. Here, we review mouse models that incorporate mutations of ASD risk genes causing pathologies in the cerebellum and striatum and highlight the vulnerability of related circuit dysfunctions within these brain regions in ASD pathophysiology.

Dysfunctional cerebellar Purkinje cells contribute to autism-like behaviour in Shank2-deficient mice.

Loss-of-function mutations in the gene encoding the postsynaptic scaffolding protein SHANK2 are a highly penetrant cause of autism spectrum disorders (ASD) involving cerebellum-related motor problems. Recent studies have implicated cerebellar pathology in the aetiology of ASD. Here we evaluate the possibility that cerebellar Purkinje cells (PCs) represent a critical locus of ASD-like pathophysiology in mice lacking Shank2. Absence of Shank2 impairs both PC intrinsic plasticity and induction of long-term potentiation at the parallel fibre to PC synapse. Moreover, inhibitory input onto PCs is significantly enhanced, most prominently in the posterior lobe where simple spike (SS) regularity is most affected. Using PC-specific Shank2 knockouts, we replicate alterations of SS regularity in vivo and establish cerebellar dependence of ASD-like behavioural phenotypes in motor learning and social interaction. These data highlight the importance of Shank2 for PC function, and support a model by which cerebellar pathology is prominent in certain forms of ASD.

Synaptic profiles during neurite extension, refinement and retraction in the developing cochlea.

BACKGROUND: During development, excess synapses form between the central and peripheral nervous systems that are then eliminated to achieve correct connectivity. In the peripheral auditory system, the developing type I spiral ganglion afferent fibres undergo a dramatic re-organisation, initially forming connections with both sensory inner hair cells (IHCs) and outer hair cells (OHCs). The OHC connections are then selectively eliminated, leaving sparse innervation by type II afferent fibres, whilst the type I afferent synapses with IHCs are consolidated. RESULTS: We examined the molecular makeup of the synaptic contacts formed onto the IHCs and OHCs during this period of afferent fibre remodelling. We observed that presynaptic ribbons initially form at all the afferent neurite contacts, i.e. not only at the expected developing IHC-type I fibre synapses but also at OHCs where type I fibres temporarily contact. Moreover, the transient contacts forming onto OHCs possess a broad set of pre- and postsynaptic proteins, suggesting that functional synaptic connections are formed prior to the removal of type I fibre innervation. AMPA-type glutamate receptor subunits were transiently observed at the base of the OHCs, with their downregulation occurring in parallel with the withdrawal of type I fibres, dispersal of presynaptic ribbons, and downregulation of the anchoring proteins Bassoon and Shank. Conversely, at developing type I afferent IHC synapses, the presence of pre- and postsynaptic scaffold proteins was maintained, with differential plasticity in AMPA receptor subunits observed and AMPA receptor subunit composition changing around hearing onset. CONCLUSIONS: Overall our data show a differential balance in the patterns of synaptic proteins at developing afferent IHC versus OHC synapses that likely reflect their stable versus transient fates.