Sound-evoked adenosine release in cooperation with neuromodulatory circuits permits auditory cortical plasticity and perceptual learning.

Meaningful auditory memories are formed in adults when acoustic information is delivered to the auditory cortex during heightened states of attention, vigilance, or alertness, as mediated by neuromodulatory circuits. Here, we identify that, in awake mice, acoustic stimulation triggers auditory thalamocortical projections to release adenosine, which prevents cortical plasticity (i.e., selective expansion of neural representation of behaviorally relevant acoustic stimuli) and perceptual learning (i.e., experience-dependent improvement in frequency discrimination ability). This sound-evoked adenosine release (SEAR) becomes reduced within seconds when acoustic stimuli are tightly paired with the activation of neuromodulatory (cholinergic or dopaminergic) circuits or periods of attentive wakefulness. If thalamic adenosine production is enhanced, then SEAR elevates further, the neuromodulatory circuits are unable to sufficiently reduce SEAR, and associative cortical plasticity and perceptual learning are blocked. This suggests that transient low-adenosine periods triggered by neuromodulatory circuits permit associative cortical plasticity and auditory perceptual learning in adults to occur.

Innate frequency-discrimination hyperacuity in Williams-Beuren syndrome mice.

Williams-Beuren syndrome (WBS) is a rare disorder caused by hemizygous microdeletion of ∼27 contiguous genes. Despite neurodevelopmental and cognitive deficits, individuals with WBS have spared or enhanced musical and auditory abilities, potentially offering an insight into the genetic basis of auditory perception. Here, we report that the mouse models of WBS have innately enhanced frequency-discrimination acuity and improved frequency coding in the auditory cortex (ACx). Chemogenetic rescue showed frequency-discrimination hyperacuity is caused by hyperexcitable interneurons in the ACx. Haploinsufficiency of one WBS gene, Gtf2ird1, replicated WBS phenotypes by downregulating the neuropeptide receptor VIPR1. VIPR1 is reduced in the ACx of individuals with WBS and in the cerebral organoids derived from human induced pluripotent stem cells with the WBS microdeletion. Vipr1 deletion or overexpression in ACx interneurons mimicked or reversed, respectively, the cellular and behavioral phenotypes of WBS mice. Thus, the Gtf2ird1-Vipr1 mechanism in ACx interneurons may underlie the superior auditory acuity in WBS.

The NALCN channel regulates metastasis and nonmalignant cell dissemination.

We identify the sodium leak channel non-selective protein (NALCN) as a key regulator of cancer metastasis and nonmalignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric, intestinal or pancreatic adenocarcinomas in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice with gadolinium-a NALCN channel blocker-similarly increased CTCs and metastases. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium, and kidney glomeruli and tubules. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumorigenesis and unmasking a potential new target for antimetastatic therapies.

Schizophrenia-related microdeletion causes defective ciliary motility and brain ventricle enlargement via microRNA-dependent mechanisms in mice.

Progressive ventricular enlargement, a key feature of several neurologic and psychiatric diseases, is mediated by unknown mechanisms. Here, using murine models of 22q11-deletion syndrome (22q11DS), which is associated with schizophrenia in humans, we found progressive enlargement of lateral and third ventricles and deceleration of ciliary beating on ependymal cells lining the ventricular walls. The cilia-beating deficit observed in brain slices and in vivo is caused by elevated levels of dopamine receptors (Drd1), which are expressed in motile cilia. Haploinsufficiency of the microRNA-processing gene Dgcr8 results in Drd1 elevation, which is brought about by a reduction in Drd1-targeting microRNAs miR-382-3p and miR-674-3p. Replenishing either microRNA in 22q11DS mice normalizes ciliary beating and ventricular size. Knocking down the microRNAs or deleting their seed sites on Drd1 mimicked the cilia-beating and ventricular deficits. These results suggest that the Dgcr8-miR-382-3p/miR-674-3p-Drd1 mechanism contributes to deceleration of ciliary motility and age-dependent ventricular enlargement in 22q11DS.

Rejuvenation of plasticity in the brain: opening the critical period.

Cortical circuits are particularly sensitive to incoming sensory information during well-defined intervals of postnatal development called 'critical periods'. The critical period for cortical plasticity closes in adults, thus restricting the brain's ability to indiscriminately store new sensory information. For example, children acquire language in an exposure-based manner, whereas learning language in adulthood requires more effort and attention. It has been suggested that pairing sounds with the activation of neuromodulatory circuits involved in attention reopens this critical period. Here, we review two critical period hypotheses related to neuromodulation: cortical disinhibition and thalamic adenosine. We posit that these mechanisms co-regulate the critical period for auditory cortical plasticity. We also discuss ways to reopen this period and rejuvenate cortical plasticity in adults.

Restoring auditory cortex plasticity in adult mice by restricting thalamic adenosine signaling.

Circuits in the auditory cortex are highly susceptible to acoustic influences during an early postnatal critical period. The auditory cortex selectively expands neural representations of enriched acoustic stimuli, a process important for human language acquisition. Adults lack this plasticity. Here we show in the murine auditory cortex that juvenile plasticity can be reestablished in adulthood if acoustic stimuli are paired with disruption of ecto-5'-nucleotidase-dependent adenosine production or A1-adenosine receptor signaling in the auditory thalamus. This plasticity occurs at the level of cortical maps and individual neurons in the auditory cortex of awake adult mice and is associated with long-term improvement of tone-discrimination abilities. We conclude that, in adult mice, disrupting adenosine signaling in the thalamus rejuvenates plasticity in the auditory cortex and improves auditory perception.

Specific disruption of thalamic inputs to the auditory cortex in schizophrenia models.

Auditory hallucinations in schizophrenia are alleviated by antipsychotic agents that inhibit D2 dopamine receptors (Drd2s). The defective neural circuits and mechanisms of their sensitivity to antipsychotics are unknown. We identified a specific disruption of synaptic transmission at thalamocortical glutamatergic projections in the auditory cortex in murine models of schizophrenia-associated 22q11 deletion syndrome (22q11DS). This deficit is caused by an aberrant elevation of Drd2 in the thalamus, which renders 22q11DS thalamocortical projections sensitive to antipsychotics and causes a deficient acoustic startle response similar to that observed in schizophrenic patients. Haploinsufficiency of the microRNA-processing gene Dgcr8 is responsible for the Drd2 elevation and hypersensitivity of auditory thalamocortical projections to antipsychotics. This suggests that Dgcr8-microRNA-Drd2-dependent thalamocortical disruption is a pathogenic event underlying schizophrenia-associated psychosis.

Thalamocortical long-term potentiation becomes gated after the early critical period in the auditory cortex.

Cortical maps in sensory cortices are plastic, changing in response to sensory experience. The cellular site of such plasticity is currently debated. Thalamocortical (TC) projections deliver sensory information to sensory cortices. TC synapses are currently dismissed as a locus of cortical map plasticity because TC synaptic plasticity is thought to be limited to neonates, whereas cortical map plasticity can be induced in both neonates and adults. However, in the auditory cortex (ACx) of adults, cortical map plasticity can be induced if animals attend to a sound or receive sounds paired with activation of cholinergic inputs from the nucleus basalis. We now show that, in the ACx, long-term potentiation (LTP), a major form of synaptic plasticity, is expressed at TC synapses in both young and mature mice but becomes gated with age. Using single-cell electrophysiology, two-photon glutamate uncaging, and optogenetics in TC slices containing the auditory thalamus and ACx, we show that TC LTP is expressed postsynaptically and depends on group I metabotropic glutamate receptors. TC LTP in mature ACx can be unmasked by cortical disinhibition combined with activation of cholinergic inputs from the nucleus basalis. Cholinergic inputs passing through the thalamic radiation activate M1 muscarinic receptors on TC projections and sustain glutamate release at TC synapses via negative regulation of presynaptic adenosine signaling through A1 adenosine receptors. These data indicate that TC LTP in the ACx persists throughout life and therefore can potentially contribute to experience-dependent cortical map plasticity in the ACx in both young and adult animals.

Presynaptic gating of postsynaptic synaptic plasticity: a plasticity filter in the adult auditory cortex.

Sensory cortices can not only detect and analyze incoming sensory information but can also undergo plastic changes while learning behaviorally important sensory cues. This experience-dependent cortical plasticity is essential for shaping and modifying neuronal circuits to perform computations of multiple, previously unknown sensations, the adaptive process that is believed to underlie perceptual learning. Intensive efforts to identify the mechanisms of cortical plasticity have provided several important clues; however, the exact cellular sites and mechanisms within the intricate neuronal networks that underlie cortical plasticity have yet to be elucidated. In this review, we present several parallels between cortical plasticity in the auditory cortex and recently discovered mechanisms of synaptic plasticity gating at thalamocortical projections that provide the main input to sensory cortices. Striking similarities between the features and mechanisms of thalamocortical synaptic plasticity and those of experience-dependent cortical plasticity in the auditory cortex, especially in terms of regulation of an early critical period, point to thalamocortical projections as an important locus of plasticity in sensory cortices.

FMRP regulates neurotransmitter release and synaptic information transmission by modulating action potential duration via BK channels.

Loss of FMRP causes fragile X syndrome (FXS), but the physiological functions of FMRP remain highly debatable. Here we show that FMRP regulates neurotransmitter release in CA3 pyramidal neurons by modulating action potential (AP) duration. Loss of FMRP leads to excessive AP broadening during repetitive activity, enhanced presynaptic calcium influx, and elevated neurotransmitter release. The AP broadening defects caused by FMRP loss have a cell-autonomous presynaptic origin and can be acutely rescued in postnatal neurons. These presynaptic actions of FMRP are translation independent and are mediated selectively by BK channels via interaction of FMRP with BK channel's regulatory ß4 subunits. Information-theoretical analysis demonstrates that loss of these FMRP functions causes marked dysregulation of synaptic information transmission. FMRP-dependent AP broadening is not limited to the hippocampus, but also occurs in cortical pyramidal neurons. Our results thus suggest major translation-independent presynaptic functions of FMRP that may have important implications for understanding FXS neuropathology.

Presynaptic gating of postsynaptically expressed plasticity at mature thalamocortical synapses.

Thalamocortical (TC) projections provide the major pathway for ascending sensory information to the mammalian neocortex. Arrays of these projections form synaptic inputs on thalamorecipient neurons, thus contributing to the formation of receptive fields (RFs) in sensory cortices. Experience-dependent plasticity of RFs persists throughout an organism's life span but in adults requires activation of cholinergic inputs to the cortex. In contrast, synaptic plasticity at TC projections is limited to the early postnatal period. This disconnect led to the widespread belief that TC synapses are the principal site of RF plasticity only in neonatal sensory cortices, but that they lose this plasticity upon maturation. Here, we tested an alternative hypothesis that mature TC projections do not lose synaptic plasticity but rather acquire gating mechanisms that prevent the induction of synaptic plasticity. Using whole-cell recordings and direct measures of postsynaptic and presynaptic activity (two-photon glutamate uncaging and two-photon imaging of the FM 1-43 assay, respectively) at individual synapses in acute mouse brain slices that contain the auditory thalamus and cortex, we determined that long-term depression (LTD) persists at mature TC synapses but is gated presynaptically. Cholinergic activation releases presynaptic gating through M(1) muscarinic receptors that downregulate adenosine inhibition of neurotransmitter release acting through A(1) adenosine receptors. Once presynaptic gating is released, mature TC synapses can express LTD postsynaptically through group I metabotropic glutamate receptors. These results indicate that synaptic plasticity at TC synapses is preserved throughout the life span and, therefore, may be a cellular substrate of RF plasticity in both neonate and mature animals.

Impaired locomotor learning and altered cerebellar synaptic plasticity in pep-19/PCP4-null mice.

PEP-19/PCP4 maps within the Down syndrome critical region and encodes a small, predominantly neuronal, IQ motif protein. Pep-19 binds calmodulin and inhibits calmodulin-dependent signaling, which is critical for synaptic function, and therefore alterations in Pep-19 levels may affect synaptic plasticity and behavior. To investigate its possible role, we generated and characterized pep-19/pcp4-null mice. Synaptic plasticity at excitatory synapses of cerebellar Purkinje cells, which express the highest levels of Pep-19, was dramatically altered in pep-19/pcp4-null mice. Instead of long-term depression, pep-19/pcp4-null mice exhibited long-term potentiation at parallel fiber-Purkinje cell synapses. The mutant mice have a marked deficit in their ability to learn a locomotor task, as measured by improved performance upon repeated testing on an accelerating rotarod. Thus, our data indicate that pep-19/pcp4 is a critical determinant of synaptic plasticity in cerebellum and locomotor learning.

Connectivity patterns revealed by mapping of active inputs on dendrites of thalamorecipient neurons in the auditory cortex.

Despite being substantially outnumbered by intracortical inputs on thalamorecipient neurons, thalamocortical projections efficiently deliver acoustic information to the auditory cortex. We hypothesized that thalamic projections may achieve effectiveness by forming synapses at optimal locations on dendritic trees of cortical neurons. Using two-photon calcium imaging in dendritic spines, we constructed maps of active thalamic and intracortical inputs on dendritic trees of thalamorecipient cortical neurons in mouse thalamocortical slices. These maps revealed that thalamic projections synapse preferentially on stubby dendritic spines within 100 microm of the soma, whereas the locations and morphology of spines that receive intracortical projections have a less-defined pattern. Using two-photon photolysis of caged glutamate, we found that activation of stubby dendritic spines located perisomatically generated larger postsynaptic potentials in the soma of thalamorecipient neurons than did activation of remote dendritic spines or spines of other morphological types. These results suggest a novel mechanism of reliability of thalamic projections: the positioning of crucial afferent inputs at optimal synaptic locations.

Dissecting the components of long-term potentiation.

The formation of memories relies in part on plastic changes at synapses between neurons. Although the mechanisms of synaptic plasticity have been studied extensively over several decades, many aspects of this process remain controversial. The cellular locus of expression of long-term potentiation (LTP), a major form of synaptic plasticity, is one of the most important unresolved phenomena. In this article, some recent advances in this area made possible by the development of new imaging tools are summarized. These studies have demonstrated that LTP is compound in nature and consists of both presynaptic and postsynaptic components. Some features of presynaptic and postsynaptic changes during compound LTP are also reviewed.

Modulation of Kv4.2 K+ currents by neuronal interleukin-16, a PDZ domain-containing protein expressed in the hippocampus and cerebellum.

Neuronal interleukin-16 (NIL-16) is a multi-PDZ domain protein expressed in post-mitotic neurons of the hippocampus and cerebellum. NIL-16 contains four PDZ domains, two of which are located within the neuron-specific N-terminal region. In yeast two-hybrid systems, the N-terminus of NIL-16 interacts with several ion channel proteins, including the Kv4.2 subunit of A-type K(+) channels. Here we provide evidence that NIL-16, through interactions with Kv4.2, influences Kv4.2 channel function and subcellular distribution. Specifically, coexpression of NIL-16 with Kv4.2 in COS-7 cells results in a significant reduction in whole-cell A-type current densities; however, when the Kv4.2 PDZ-ligand domain is mutated, Kv4.2 current densities are not affected by NIL-16 coexpression. Moreover, single-channel conductance was not influenced by the presence of NIL-16. In hippocampal neurons, A-type current densities are increased by conditions that inhibit interactions between NIL-16 and Kv4.2, such as overexpression of the Kv4.2 C-terminal PDZ-ligand domain and treatment with small-interfering RNA duplexes that reduce NIL-16 expression. Results of surface biotinylation assays using COS-7 cells suggest that Kv4.2 surface expression levels are reduced by coexpression with NIL-16. In addition, coexpression of NIL-16 with Kv4.2 induces Kv4.2 to form dense intracellular clusters; whereas without NIL-16, Kv4.2 channels cells are dispersed. Taken together, these data suggest that interactions between Kv4.2 and NIL-16 may reduce the number of functional Kv4.2-containing channels on the cell surface. In summary, NIL-16 may provide a novel form of A-type K(+) channel modulation that is localized specifically to neurons of the hippocampus and cerebellum.

A biochemical assay for acetylcholinesterase activity in PC12 cells.

This lab describes two biochemical assays: One for measuring acetylcholinesterase activity and one for measuring protein concentration. Students learn how to manipulate small-volume samples, use a standard spectrophotometer or a microplate reader spectrophotometer, construct a standard curve, and normalize data. The lab is intended to be used in conjunction with a cell culture lab in which PC12 cells are exposed to various agents that influence their phenotypic state.

Differentiation of PC12 cells.

This 3-week-long series of collaborative laboratory exercises explores how to use a cultured cell system (PC12 cells) to study signaling pathways involved in cellular differentiation. The laboratory would be useful in a neurobiology or cell biology course for advanced undergraduate students. The background and details for performing the lab are provided along with suggestions for assessing student performance and understanding.