Neurons In Mutant Mice Research Articles

Disrupted Choline Clearance and Sustained Acetylcholine Release In Vivo by a Common Choline Transporter Coding Variant Associated with Poor Attentional Control in Humans.

Transport of choline via the neuronal high-affinity choline transporter (CHT; SLC5A7) is essential for cholinergic terminals to synthesize and release acetylcholine (ACh). In humans, we previously demonstrated an association between a common CHT coding substitution (rs1013940; Ile89Val) and reduced attentional control as well as attenuated frontal cortex activation. Here, we used a CRISPR/Cas9 approach to generate mice expressing the I89V substitution and assessed, in vivo, CHT-mediated choline transport, and ACh release. Relative to wild-type (WT) mice, CHT-mediated clearance of choline in male and female mice expressing one or two Val89 alleles was reduced by over 80% in cortex and over 50% in striatum. Choline clearance in CHT Val89 mice was further reduced by neuronal inactivation. Deficits in ACh release, 5 and 10 min after repeated depolarization at a low, behaviorally relevant frequency, support an attenuated reloading capacity of cholinergic neurons in mutant mice. The density of CHTs in total synaptosomal lysates and neuronal plasma-membrane-enriched fractions was not impacted by the Val89 variant, indicating a selective impact on CHT function. When challenged with a visual disruptor to reveal attentional control mechanisms, Val89 mice failed to adopt a more conservative response bias. Structural modeling revealed that Val89 may attenuate choline transport by altering conformational changes of CHT that support normal transport rates. Our findings support the view that diminished sustained cholinergic signaling capacity underlies perturbed attentional performance in individuals expressing CHT Val89. The CHT Val89 mouse serves as a valuable model to study heritable risk for cognitive disorders arising from cholinergic dysfunction.SIGNIFICANCE STATEMENT Acetylcholine (ACh) signaling depends on the functional capacity of the neuronal choline transporter (CHT). Previous research demonstrated that humans expressing the common CHT coding variant Val89 exhibit attentional vulnerabilities and attenuated fronto-cortical activation during attention. Here, we find that mice engineered to express the Val89 variant exhibit reduced CHT-mediated choline clearance and a diminished capacity to sustain ACh release. Additionally, Val89 mice lack cognitive flexibility in response to an attentional challenge. These findings provide a mechanistic and cognitive framework for interpreting the attentional phenotype associated with the human Val89 variant and establish a model that permits a more invasive interrogation of CNS effects as well as the development of therapeutic strategies for those, including Val89 carriers, with presynaptic cholinergic perturbations.

Optogenetic Activation of Striatopallidal Neurons Reveals Altered HCN Gating in DYT1 Dystonia

Firing activity of external globus pallidus (GPe) is crucial for motor control and is severely perturbed in dystonia, a movement disorder characterized by involuntary, repetitive muscle contractions. Here, we show that GPe projection neurons exhibit a reduction of firing frequency and an irregular pattern in a DYT1 dystonia model. Optogenetic activation of the striatopallidal pathway fails to reset pacemaking activity of GPe neurons in mutant mice. Abnormal firing is paralleled by alterations in motor learning. We find that loss of dopamine D2 receptor-dependent inhibition causes increased GABA input at striatopallidal synapses, with subsequent downregulation of hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels. Accordingly, enhancing invivo HCN channel activity or blocking GABA release restores both the ability of striatopallidal inputs to pause ongoing GPe activity and motor coordination deficits. Our findings demonstrate an impaired striatopallidal connectivity, supporting the central role of GPe in motor control and, more importantly, identifying potential pharmacological targets for dystonia.

Functional and Structural Impairments in the Perirhinal Cortex of a Mouse Model of CDKL5 Deficiency Disorder Are Rescued by a TrkB Agonist.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodevelopmental encephalopathy caused by mutations in the CDKL5 gene and characterized by early-onset epilepsy and intellectual and motor impairments. No cure is currently available for CDD patients, as limited knowledge of the pathology has hindered the development of therapeutics. Cdkl5 knockout (KO) mouse models, recently created to investigate the role of CDKL5 in the etiology of CDD, recapitulate various features of the disorder. Previous studies have shown alterations in synaptic plasticity and dendritic pattern in the cerebral cortex and in the hippocampus, but the knowledge of the molecular substrates underlying these alterations is still limited. Here, we have examined for the first time synaptic function and plasticity, dendritic morphology, and signal transduction pathways in the perirhinal cortex (PRC) of this mouse model. Being interconnected with a wide range of cortical and subcortical structures and involved in various cognitive processes, PRC provides a very interesting framework for examining how CDKL5 mutation leads to deficits at the synapse, circuit, and behavioral level. We found that long-term potentiation (LTP) was impaired, and that the TrkB/PLCγ1 pathway could be mechanistically involved in this alteration. PRC neurons in mutant mice showed a reduction in dendritic length, dendritic branches, PSD-95-positive puncta, GluA2-AMPA receptor levels, and spine density and maturation. These functional and structural deficits were associated with impairment in visual recognition memory. Interestingly, an in vivo treatment with a TrkB agonist (the 7,8-DHF prodrug R13) to trigger the TrkB/PLCγ1 pathway rescued defective LTP, dendritic pattern, PSD-95 and GluA2-AMPA receptor levels, and restored visual recognition memory in Cdkl5 KO mice. Present findings demonstrate a critical role of TrkB signaling in the synaptic development alterations due to CDKL5 mutation, and suggest the possibility of TrkB-targeted pharmacological interventions.

Circuit level defects in the developing neocortex of Fragile X mice

Subtle alterations in how cortical network dynamics are modulated by different behavioral states could disrupt normal brain function and underlie symptoms of neuropsychiatric disorders, including fragile X syndrome (FXS). Using two-photon calcium imaging and electrophysiology we recorded spontaneous neuronal ensemble activity in mouse somatosensory cortex. Unanesthetized Fmr1–/– mice exhibited abnormally high synchrony of neocortical network activity, especially during the first two postnatal weeks. Neuronal firing rates were 3-fold higher in Fmr1–/– mice during whole-cell recordings manifesting Up/Down states (slow wave sleep, quiet wakefulness), likely due to a higher firing probability during Up states. Combined EEG/calcium imaging experiments confirmed that neurons in mutant mice have abnormally high firing and synchrony during sleep. We conclude that cortical networks in FXS are hyperexcitable in a brain state-dependent manner during a critical period for experience-dependent plasticity. These state-dependent network defects could explain the intellectual, sleep and sensory integration dysfunctions associated with FXS.

Subtle microstructural changes of the striatum in a DYT1 knock-in mouse model of dystonia

The dystonias are comprised of a group of disorders that share common neurological abnormalities of involuntary twisting or repetitive movements and postures. The most common inherited primary dystonia is DYT1 dystonia, which is due to loss of a GAG codon in the TOR1A gene that encodes torsinA. Autopsy studies of brains from patients with DYT1 dystonia have revealed few abnormalities, although recent neuroimaging studies have implied the existence of microstructural defects that might not be detectable with traditional histopathological methods. The current studies took advantage of a knock-in mouse model for DYT1 dystonia to search for subtle anatomical abnormalities in the striatum, a region often implicated in studies of dystonia. Multiple abnormalities were identified using a combination of quantitative stereological measures of immunohistochemical stains for specific neuronal populations, morphometric studies of Golgi-stained neurons, and immuno-electron microscopy of synaptic connectivity. In keeping with other studies, there was no obvious loss of striatal neurons in the DYT1 mutant mice. However, interneurons immunoreactive for choline acetyltransferase or parvalbumin were larger in the mutants than in control mice. In contrast, interneurons immunoreactive for neuronal nitric oxide synthase were smaller in the mutants than in controls. Golgi histochemical studies of medium spiny projection neurons in the mutant mice revealed slightly fewer and thinner dendrites, and a corresponding loss of dendritic spines. Electron microscopic studies showed a reduction in the ratio of axo-spinous to axo-dendritic synaptic inputs from glutamatergic and dopaminergic sources in mutant mice compared with controls. These results suggest specific anatomical substrates for altered signaling in the striatum and potential correlates of the abnormalities implied by human imaging studies of DYT1 dystonia.

δ-Catenin Is Required for the Maintenance of Neural Structure and Function in Mature Cortex In Vivo

Delta-catenin is a brain-specific member of the adherens junction complex that localizes to the postsynaptic and dendritic compartments. This protein is likely critical for normal cognitive function; its hemizygous loss is linked to the severe mental retardation syndrome Cri-du-Chat and it directly interacts with presenilin-1 (PS1), the protein most frequently mutated in familial Alzheimer's disease. Here we examine dendritic structure and cortical function in vivo in mice lacking delta-catenin. We find that in cerebral cortex of 5-week-old mice, dendritic complexity, spine density, and cortical responsiveness are similar between mutant and littermate controls; thereafter, mutant mice experience progressive dendritic retraction, a reduction in spine density and stability, and concomitant reductions in cortical responsiveness. Our results indicate that delta-catenin regulates the maintenance of dendrites and dendritic spines in mature cortex but does not appear to be necessary for the initial establishment of these structures during development.

Comparison of γ-aminobutyrate receptors in the medial vestibular nucleus of control and Scn8a mutant mice

The Purkinje cells of the cerebellum provide inhibitory input to vestibular nucleus neurons, with γ-aminobutyrate (GABA) as neurotransmitter. Using extracellular recordings and bath application of agonists and antagonists, we compared GABA receptors in the medial vestibular nucleus of brain slices from Scn8a mutant mice of med J type, in which there is greatly reduced spontaneous and evoked activity of Purkinje cells, to those in slices from control mice. Muscimol, an agonist at GABA A receptors, produced a larger reduction of firing rate in neurons of mutant mice than in neurons of control mice, whereas there was no difference for baclofen, an agonist at GABA B receptors. In most cases tested, the effects of muscimol and baclofen remained similar when synaptic transmission was blocked, suggesting that the effects were predominantly directly upon GABA receptors of the neurons being recorded from. The up-regulation of GABA A receptors was similar in magnitude to that previously found for rats with bilateral transection of the inferior cerebellar peduncle. It may relate in both cases to reduced Purkinje cell input to medial vestibular nucleus neurons. The lack of effect on GABA B receptors suggests that the changes found with peduncle transection may have resulted from something more than reduced Purkinje cell activity, such as reduced concentrations of GABA, or that reduction of Purkinje cell activity in Scn8a mutant mice was insufficient to affect GABA B receptors. Other possible explanations of the results cannot be excluded since the Scn8a mutation affects other neuron types besides Purkinje cells.

Bcl‐2 overexpression delays caspase‐3 activation and rescues cerebellar degeneration in prion‐deficient mice that overexpress amino‐terminally truncated prion

Prnp knockout mice that overexpress an amino-truncated form of PrPc (deltaPrP) are ataxic and display cerebellar cell loss and premature death. Studies on the molecular and intracellular events that trigger cell death in these mutants may contribute to elucidate the functions of PrPc and to the design of treatments for prion disease. Here we examined the effects of Bcl-2 overexpression in neurons on the development of the neurological syndrome and cerebellar pathology of deltaPrP. We show that deltaPrP overexpression activates the stress-associated kinases ERK1-2 in reactive astroglia, p38 and the phosphorylation of p53, which leads to the death of cerebellar neurons in mutant mice. We found that the expression of deltaPrP in cell lines expressing very low levels of PrPc strongly induces the activation of apoptotic pathways, thereby leading to caspase-3 activation and cell death, which can be prevented by coexpressing Bcl-2. Finally, we corroborate in vivo that neuronal-directed Bcl-2 overexpression in deltaPrP mice (deltaPrP Bcl-2) markedly reduces caspase-3 activation, glial activation, and neuronal cell death in cerebellum by improving locomotor deficits and life expectancy.

Differential contribution of TRPV1 to thermal responses and tissue injury-induced sensitization of dorsal horn neurons in laminae I and V in the mouse

Our previous recordings from dorsal root ganglion and spinal lamina V neurons from TRPV1-mutant mice showed dramatic decreases in responses to temperatures near the activation threshold of this channel (43–49 °C). Somewhat unexpectedly, we only observed behavioral deficits in these mice at higher temperatures (50–58 °C). In the present study, we tested the hypothesis that the noxious heat-evoked pain behavior that persists in TRPV1-mutant mice reflects residual responsiveness of neurons in the superficial, but not deep, dorsal horn. To this end, we performed in vivo extracellular recordings of spinal nociresponsive neurons in laminae I and V in wild type (WT) and TRPV1 mutant mice. Neurons in WT and mutant mice from both laminae did not differ in their spontaneous activity or evoked responses to mechanical or cold stimuli. By contrast, most lamina I neurons from mutant mice responded to noxious heat with significantly higher thresholds than in WT mice. In contrast, lamina V neurons from mutant mice were virtually unresponsive to noxious heat before and after topical mustard oil-induced tissue injury. Interestingly, lamina I neurons in mutant mice displayed thermal sensitization following tissue injury, comparable in magnitude, but of shorter duration, than in WT mice. We conclude that TRPV1 is necessary for noxious heat-evoked responses of lamina V neurons, both before and after tissue injury. It is also an essential contributor to the normal activation threshold of lamina I neurons to noxious heat and for the full duration of thermal sensitization of lamina I neurons following injury. Finally, our results suggest that the processing of noxious thermal messages by neurons in lamina I involves convergent inputs from a heterogeneous population of primary afferent thermal nociceptors.

NSCL-1 and NSCL-2 synergistically determine the fate of GnRH-1 neurons and control necdin gene expression.

To study the role of the bHLH genes NSCL-1 and NSCL-2 in the development of GnRH-1 neurons, we have generated compound mutant mice. Mutant animals die at birth and show a virtually complete absence of GnRH-1 neurons in the posterior parts of the brain at E18.5 and an aberrant morphology of the remaining GnRH-1 neurons in the anterior parts of the brain indicating that NSCL-1 and NSCL-2 might concomitantly control differentiation/migration of GnRH-1 neurons in a cell autonomous manner. To gain further insights into this process, we screened for NSCL target genes using DNA array hybridization and detected necdin, which is deleted in the human Prader-Willi syndrome phenotypically resembling the NSCL-2 mutation. Using chromatin immunoprecipitation and site-directed mutagenesis of the necdin promoter, we demonstrate that NSCLs together with additional cofactors directly control transcription of the necdin gene. NSCL-dependent control of necdin expression might be instrumental for proper neuronal cell differentiation and enable GnRH-1 neurons to migrate.

Electrophysiological characteristics of inferior colliculus neurons in mutant mice with hereditary absence of cochlear outer hair cells

One strain of homozygous Kit W-v mice (formerly known as W v/W v) lack 98% of the cochlear outer hair cells (OHCs) from birth. Inner hair cells (IHCs) and supporting cells develop normally. Thus, this strain is an attractive model to study the effect of complete OHC absence on central frequency representation. Frequency threshold curves were recorded along the tonotopic axis of inferior colliculus (IC) in mutant and control mice of the genetic background strain (C57BL/6J) and a different outbred strain (NMRI/wild mouse hybrids) known to be free of any cochlear pathology. The average threshold level of neurons in the mutants was 100 dB sound pressure level, 60 dB higher than in C57BL/6J and NMRI mice. Their tuning curves lacked the sharply tuned tip. In the C57BL/6J mice, although younger than four months, abnormal tuning curves were found for about 30% of the neurons, especially in the high frequency range. No abnormal tuning curves were found in the NMRI mice. The bandwidth of the tuning curves, measured at 10 dB above threshold, was on average 1.27 octaves in mutants, 0.62 octaves in C57BL/6J mice, and 0.34 octaves in NMRI mice. The range for the high cut-off frequency of the tuning curves at 10 dB above threshold was 6.4–61.1 kHz in the NMRI and 7–59.5 kHz in C57BL/6J. In the mutants, the range was limited to 11.1–41.7 kHz. The tonotopic gradient based on the cut-off frequency was less steep in the IC of the mutants than in both control groups.

Increased neuronal excitability and seizures in the Na(+)/H(+) exchanger null mutant mouse.

Mice lacking the Na(+)/H(+) exchanger isoform 1 (NHE1) manifest neurological diseases that include ataxia, motor deficits, and a seizure disorder. The molecular basis for the phenotype has not been clear, and it has not been determined how the lack of NHE1 leads, in particular, to the seizure disorder. We have shown in this work that hippocampal CA1 neurons in mutant mice have a much higher excitability than in wild-type mice. This higher excitability is partly based on an upregulation of the Na(+) current density (608.2 +/- 123.2 pA/pF in NHE1 mutant vs. 334.7 +/- 63.7 pA/pF in wild type in HCO/CO(2)). Alterations in Na(+) channel characteristics, including steady-state inactivation (shift of 18 mV in the depolarization direction in the mutant), recovery from inactivation (tau(h) = 5.22 +/- 0.49 ms in wild-type neurons and 2.20 +/- 0.20 ms in mutant neurons), and deactivation (at -100 mV, tau(d) = 1.75 +/- 0.53 ms in mutant and 0.21 +/- 0.05 ms in wild-type neurons) further enhance the differences in excitability between mutant and wild-type mice. Our investigation demonstrates the existence of an important functional interaction between the NHE1 protein and the voltage-sensitive Na(+) channel. We hypothesize that the increased neuronal excitability and possibly the seizure disorder in mice lacking the NHE1 is due, at least in part, to changes in Na(+) channel expression and/or regulation.

Pituitary hormones as neurotrophic signals: anomalous hypophysiotrophic neuron differentiation in hypopituitary dwarf mice.

Anterior pituitary hormones are known to exert dynamic negative feedback effects on their respective regulatory ("hypophysiotropic") neurons in the hypothalamus. The purpose of this review is to present the evidence for a theory that the effect of pituitary hormones on these hypophysiotropic neurons is neurotrophic, extending beyond dynamic feedback to influence upon cell survival, phenotypic differentiation, and axonal connectivity. To that end, the adult condition and the development of hypophysiotropic neurons in mutant mice which lack pituitary growth hormone (GH) and prolactin (PRL) are presented as models of the effect of absent specific neurotrophic signals. The expression of the neurohormones which inhibit PRL and GH secretion, dopamine (DA) and somatostatin, respectively, is markedly reduced in the hypothalamus of the hypopituitary dwarf mouse, and this adult condition is the result of postnatal failure to develop or actual regression, which may include neuronal cell death. The deficit in DA may be reversed by PRL replacement, but only if initiated at an identified critical postnatal period. Conversely, expression of the stimulatory GH-releasing hormone (GHRH) is markedly increased in the dwarf mouse hypothalamus. The loss of DA and the increase in GHRH occur in the same hypothalamic area, suggesting neuronal phenotypic plasticity in response to absence of pituitary feedback signals. The axonal terminations of extant GH- and PRL-regulating neurons in external median eminence appear to be reduced, suggesting that pituitary signals are required for appropriate axonal guidance during development, even though an endocrine vascular route intervenes between these regulatory neurons and their target secretory cells. The collective observations indicate that GH and PRL may be regarded as neurotrophic factors for their respective regulatory neurons in the hypothalamus.