Detlef H. Heck

Consensus Paper: Pathological Role of the Cerebellum in Autism

There has been significant advancement in various aspects of scientific knowledge concerning the role of cerebellum in the etiopathogenesis of autism. In the current consensus paper, we will observe the diversity of opinions regarding the involvement of this important site in the pathology of autism. Recent emergent findings in literature related to cerebellar involvement in autism are discussed, including: cerebellar pathology, cerebellar imaging and symptom expression in autism, cerebellar genetics, cerebellar immune function, oxidative stress and mitochondrial dysfunction, GABAergic and glutamatergic systems, cholinergic, dopaminergic, serotonergic, and oxytocin-related changes in autism, motor control and cognitive deficits, cerebellar coordination of movements and cognition, gene–environment interactions, therapeutics in autism, and relevant animal models of autism. Points of consensus include presence of abnormal cerebellar anatomy, abnormal neurotransmitter systems, oxidative stress, cerebellar motor and cognitive deficits, and neuroinflammation in subjects with autism. Undefined areas or areas requiring further investigation include lack of treatment options for core symptoms of autism, vermal hypoplasia, and other vermal abnormalities as a consistent feature of autism, mechanisms underlying cerebellar contributions to cognition, and unknown mechanisms underlying neuroinflammation.

Connecting the dots of the cerebro‐cerebellar role in cognitive function: Neuronal pathways for cerebellar modulation of dopamine release in the prefrontal cortex

Cerebellar involvement in autism, schizophrenia, and other cognitive disorders is typically associated with prefrontal cortical pathology. However, the underlying neuronal mechanisms are largely unknown. It has previously been shown in mice that stimulation of the dentate nucleus (DN) of the cerebellum evokes dopamine (DA) release in the medial prefrontal cortex (mPFC). Here, we investigated the neuronal circuitry by which the cerebellum modulates mPFC DA release. Fixed potential amperometry was used to determine the contribution of two candidate pathways by which the cerebellum may modulate mPFC DA release. In urethane anesthetized mice, DA release evoked by DN stimulation (50 Hz) was recorded in mPFC following local anesthetic lidocaine (0.02 μg) or ionotropic glutamate receptor antagonist kynurenate (0.5 μg) infusions into the mediodorsal or ventrolateral thalamic nucleus (ThN md; ThN vl), or the ventral tegmental area (VTA). Following intra‐VTA lidocaine or kynurenate infusions, DA release was decreased by ∼50%. Following intra‐ThN md and ThN vl infusions of either drug, DA release was decreased by ∼35% and 15%, respectively. Reductions in DA release following lidocaine or kynurenate infusions were not significantly different indicating that neuronal cells in the VTA and ThN were activated primarily if not entirely by glutamatergic inputs. The present study suggests that neuropathological changes in the cerebellum commonly observed in autism, schizophrenia, and other cognitive disorders could result in a loss of functionality of cerebellar‐mPFC circuitry that is manifested as aberrant dopaminergic activity in the mPFC. Additionally, these results specifically implicate glutamate as a modulator of mPFC dopaminergic activity. Synapse, 2011.

Comprehensive Analysis of Ultrasonic Vocalizations in a Mouse Model of Fragile X Syndrome Reveals Limited, Call Type Specific Deficits

Fragile X syndrome (FXS) is a well-recognized form of inherited mental retardation, caused by a mutation in the fragile X mental retardation 1 (Fmr1) gene. The gene is located on the long arm of the X chromosome and encodes fragile X mental retardation protein (FMRP). Absence of FMRP in fragile X patients as well as in Fmr1 knockout (KO) mice results, among other changes, in abnormal dendritic spine formation and altered synaptic plasticity in the neocortex and hippocampus. Clinical features of FXS include cognitive impairment, anxiety, abnormal social interaction, mental retardation, motor coordination and speech articulation deficits. Mouse pups generate ultrasonic vocalizations (USVs) when isolated from their mothers. Whether those social ultrasonic vocalizations are deficient in mouse models of FXS is unknown. Here we compared isolation-induced USVs generated by pups of Fmr1-KO mice with those of their wild type (WT) littermates. Though the total number of calls was not significantly different between genotypes, a detailed analysis of 10 different categories of calls revealed that loss of Fmr1 expression in mice causes limited and call-type specific deficits in ultrasonic vocalization: the carrier frequency of flat calls was higher, the percentage of downward calls was lower and that the frequency range of complex calls was wider in Fmr1-KO mice compared to their WT littermates.

Spike timing and reliability in cortical pyramidal neurons: Effects of EPSC kinetics, input synchronization and background noise on spike timing

In vivo studies have shown that neurons in the neocortex can generate action potentials at high temporal precision. The mechanisms controlling timing and reliability of action potential generation in neocortical neurons, however, are still poorly understood. Here we investigated the temporal precision and reliability of spike firing in cortical layer V pyramidal cells at near-threshold membrane potentials. Timing and reliability of spike responses were a function of EPSC kinetics, temporal jitter of population excitatory inputs, and of background synaptic noise. We used somatic current injection to mimic population synaptic input events and measured spike probability and spike time precision (STP), the latter defined as the time window (Δt) holding 80% of response spikes. EPSC rise and decay times were varied over the known physiological spectrum. At spike threshold level, EPSC decay time had a stronger influence on STP than rise time. Generally, STP was highest (≤2.45 ms) in response to synchronous compounds of EPSCs with fast rise and decay kinetics. Compounds with slow EPSC kinetics (decay time constants>6 ms) triggered spikes at lower temporal precision (≥6.58 ms). We found an overall linear relationship between STP and spike delay. The difference in STP between fast and slow compound EPSCs could be reduced by incrementing the amplitude of slow compound EPSCs. The introduction of a temporal jitter to compound EPSCs had a comparatively small effect on STP, with a tenfold increase in jitter resulting in only a five fold decrease in STP. In the presence of simulated synaptic background activity, precisely timed spikes could still be induced by fast EPSCs, but not by slow EPSCs.

C57BL/6J and DBA/2J mice vary in lick rate and ingestive microstructure

Fluid licking in mice is an example of a rhythmic behavior thought to be under the control of a central pattern generator. Inbred strains of mice have been shown to differ in mean or modal interlick interval (ILI) duration, suggesting a genetic‐based variation. We investigated water licking in the commonly used inbred strains C57BL/6J (B6) and DBA/2J (D2), using a commercially available contact lickometer. Results from 20‐min test sessions indicated that D2 mice lick at a faster rate than B6 mice (10.6 licks/s vs. 8.5 licks/s), based on analysis of the distribution of short‐duration ILIs (50–160 ms). This strain difference was independent of sex, extent of water deprivation or total number of licks. D2 mice also displayed a faster lick rate when the strains were tested with a series of brief (5 s) trials. However, when ingestion over the entire 20‐min session was analyzed, it was evident that D2 mice had an overall slower rate of ingestion than B6 mice. This was because of the tendency for D2 mice to have more very long pauses (>30 s) between sequences of licking bursts. Overall, it appeared that D2 mice licked more efficiently, ingesting more rapidly during excursions to the spout that were fewer and farther between.

The neuronal code(s) of the cerebellum

Understanding how neurons encode information in sequences of action potentials is of fundamental importance to neuroscience. The cerebellum is widely recognized for its involvement in the coordination of movements, which requires muscle activation patterns to be controlled with millisecond precision. Understanding how cerebellar neurons accomplish such high temporal precision is critical to understanding cerebellar function. Inhibitory Purkinje cells, the only output neurons of the cerebellar cortex, and their postsynaptic target neurons in the cerebellar nuclei, fire action potentials at high, sustained frequencies, suggesting spike rate modulation as a possible code. Yet, millisecond precise spatiotemporal spike activity patterns in Purkinje cells and inferior olivary neurons have also been observed. These results and ongoing studies suggest that the neuronal code used by cerebellar neurons may span a wide time scale from millisecond precision to slow rate modulations, likely depending on the behavioral context.

Cerebellar cortical output encodes temporal aspects of rhythmic licking movements and is necessary for normal licking frequency.

Rodents consume water by performing stereotypic, rhythmic licking movements that are believed to be controlled by brainstem pattern‐generating circuits. Previous work has shown that synchronized population activity of inferior olive neurons was phase‐locked to the licking rhythm in rats, suggesting a cerebellar involvement in temporal aspects of licking behavior. However, what role the cerebellum has in licking behavior and whether licking is represented in the high‐frequency simple spike output of Purkinje cells remains unknown. We recorded Purkinje cell simple and complex spike activity in awake mice during licking, and determined the behavioral consequences of loss of cerebellar function. Mouse cerebellar cortex contained a multifaceted representation of licking behavior encoded in the simple spike activities of Purkinje cells distributed across Crus I, Crus II and lobus simplex of the right cerebellar hemisphere. Lick‐related Purkinje cell simple spike activity was modulated rhythmically, phase‐locked to the lick rhythm, or non‐rhythmically. A subpopulation of lick‐related Purkinje cells differentially represented lick interval duration in their simple spike activity. Surgical removal of the cerebellum or temporary pharmacological inactivation of the cerebellar nuclei significantly slowed the licking frequency. Fluid licking was also less efficient in mice with impaired cerebellar function, indicated by a significant decline in the volume per lick fluid intake. The gross licking movement appeared unaffected. Our results suggest a cerebellar role in modulating the frequency of the central pattern‐generating circuits controlling fluid licking and in the fine coordination of licking, while contributing little to the coordination of the gross licking movement.

A technique for stereotaxic recordings of neuronal activity in awake, head-restrained mice

Neurophysiological recordings of brain activity during behavior in awake animals have traditionally been performed in primates because of their evolutionary close relationship to humans and comparable behavioral skills. However, with properly designed behavioral tasks, many fundamental questions about how the brain controls behavior can also be addressed in small rodents. Today, the rapid progress in mouse neurogenetics, including the development of mouse models of human brain disorders, provides unique and unparalleled opportunities for the investigation of normal and pathological brain function. The development of experimental procedures for the recording of neuronal activity in awake and behaving mice is an important and necessary step towards neurophysiological investigation of normal and pathological mouse brain function. Here we describe a method for stereotaxic recordings of neuronal activity from head-restrained mice during fluid licking. Fluid licking is a natural and spontaneous behavior in rodents, which mice readily perform under head-restrained conditions. Using a head-restrained preparation allows recordings of well-isolated single units at multiple sites during repeated experimental sessions. Thus, a large number of neurons can be tested for their relationship with behavior and detailed spatial maps of behavior related neuronal activity can be generated as exemplified here with recordings from lick-related Purkinje cells in the cerebellum.

Normal social seeking behavior, hypoactivity and reduced exploratory range in a mouse model of Angelman syndrome

BackgroundAngelman syndrome (AS) is a neurogenetic disorder characterized by severe developmental delay with mental retardation, a generally happy disposition, ataxia and characteristic behaviors such as inappropriate laughter, social-seeking behavior and hyperactivity. The majority of AS cases are due to loss of the maternal copy of the UBE3A gene. Maternal Ube3a deficiency (Ube3am-/p+), as well as complete loss of Ube3a expression (Ube3am-/p-), have been reproduced in the mouse model used here.ResultsHere we asked if two characteristic AS phenotypes - social-seeking behavior and hyperactivity - are reproduced in the Ube3a deficient mouse model of AS. We quantified social-seeking behavior as time spent in close proximity to a stranger mouse and activity as total time spent moving during exploration, movement speed and total length of the exploratory path. Mice of all three genotypes (Ube3am+/p+, Ube3am-/p+, Ube3am-/p-) were tested and found to spend the same amount of time in close proximity to the stranger, indicating that Ube3a deficiency in mice does not result in increased social seeking behavior or social dis-inhibition. Also, Ube3a deficient mice were hypoactive compared to their wild-type littermates as shown by significantly lower levels of activity, slower movement velocities, shorter exploratory paths and a reduced exploratory range.ConclusionsAlthough hyperactivity and social-seeking behavior are characteristic phenotypes of Angelman Syndrome in humans, the Ube3a deficient mouse model does not reproduce these phenotypes in comparison to their wild-type littermates. These phenotypic differences may be explained by differences in the size of the genetic defect as ~70% of AS patients have a deletion that includes several other genes surrounding the UBE3A locus.

Cerebellar Zonal Patterning Relies on Purkinje Cell Neurotransmission

Cerebellar circuits are patterned into an array of topographic parasagittal domains called zones. The proper connectivity of zones is critical for motor coordination and motor learning, and in several neurological diseases cerebellar circuits degenerate in zonal patterns. Despite recent advances in understanding zone function, we still have a limited understanding of how zones are formed. Here, we focused our attention on Purkinje cells to gain a better understanding of their specific role in establishing zonal circuits. We used conditional mouse genetics to test the hypothesis that Purkinje cell neurotransmission is essential for refining prefunctional developmental zones into sharp functional zones. Our results show that inhibitory synaptic transmission in Purkinje cells is necessary for the precise patterning of Purkinje cell zones and the topographic targeting of mossy fiber afferents. As expected, blocking Purkinje cell neurotransmission caused ataxia. Using in vivo electrophysiology, we demonstrate that loss of Purkinje cell communication altered the firing rate and pattern of their target cerebellar nuclear neurons. Analysis of Purkinje cell complex spike firing revealed that feedback in the cerebellar nuclei to inferior olive to Purkinje cell loop is obstructed. Loss of Purkinje neurotransmission also caused ectopic zonal expression of tyrosine hydroxylase, which is only expressed in adult Purkinje cells when calcium is dysregulated and if excitability is altered. Our results suggest that Purkinje cell inhibitory neurotransmission establishes the functional circuitry of the cerebellum by patterning the molecular zones, fine-tuning afferent circuitry, and shaping neuronal activity.