The PNKD gene is associated with Tourette Disorder or Tic disorder in a multiplex family.

Tourette Disorder (TD) is a childhood-onset neuropsychiatric and neurodevelopmental disorder characterized by the presence of both motor and vocal tics. The genetic architecture of TD is believed to be complex and heterogeneous. Nevertheless, DNA sequence variants co-segregating with TD phenotypes within multiplex families have been identified. This report examines whole exomes of affected and unaffected individuals in a multiplex TD family to discover genes involved in the TD etiology. We performed whole exome sequencing on six out of nine members in a three-generation TD multiplex family. Putative deleterious sequence variants co-segregating with TD patients were identified by our in-house bioinformatics pipeline. Induced pluripotent stem cells (iPSCs) were generated from one unaffected and two TD affected individuals. Neurons were derived from the iPSCs and biochemical assays were conducted to evaluate possible molecular differences between affected and unaffected. A rare heterozygous nonsense mutation in PNKD was co-segregated with TD in this multiplex family. Transcript and protein levels of the PNKD long isoform were reduced in neurons derived from the individuals with TD due to the nonsense mutation, indicating nonsense-mediated mRNA decay. We demonstrated that the PNKD long isoform monomer oligomerizes with itself as well as interacts with the synaptic active zone protein RIMS1α. We concluded that reduced PNKD long isoform levels are detected in all affected individuals and we provide evidence for a mechanism whereby this might contribute to the TD phenotype.

Intellicount: High-Throughput Quantification of Fluorescent Synaptic Protein Puncta by Machine Learning.

Synapse formation analyses can be performed by imaging and quantifying fluorescent signals of synaptic markers. Traditionally, these analyses are done using simple or multiple thresholding and segmentation approaches or by labor-intensive manual analysis by a human observer. Here, we describe Intellicount, a high-throughput, fully-automated synapse quantification program which applies a novel machine learning (ML)-based image processing algorithm to systematically improve region of interest (ROI) identification over simple thresholding techniques. Through processing large datasets from both human and mouse neurons, we demonstrate that this approach allows image processing to proceed independently of carefully set thresholds, thus reducing the need for human intervention. As a result, this method can efficiently and accurately process large image datasets with minimal interaction by the experimenter, making it less prone to bias and less liable to human error. Furthermore, Intellicount is integrated into an intuitive graphical user interface (GUI) that provides a set of valuable features, including automated and multifunctional figure generation, routine statistical analyses, and the ability to run full datasets through nested folders, greatly expediting the data analysis process.

Prolonged membrane potential depolarization in cingulate pyramidal cells after digit amputation in adult rats.

The anterior cingulate cortex (ACC) plays an important role in higher brain functions including learning, memory, and persistent pain. Long-term potentiation of excitatory synaptic transmission has been observed in the ACC after digit amputation, which might contribute to plastic changes associated with the phantom pain. Here we report a long-lasting membrane potential depolarization in ACC neurons of adult rats after digit amputation in vivo. Shortly after digit amputation of the hind paw, the membrane potential of intracellularly recorded ACC neurons quickly depolarized from approximately -70 mV to approximately -15 mV and then slowly repolarized. The duration of this amputation-induced depolarization was about 40 min. Intracellular staining revealed that these neurons were pyramidal neurons in the ACC. The depolarization is activity-dependent, since peripheral application of lidocaine significantly reduced it. Furthermore, the depolarization was significantly reduced by a NMDA receptor antagonist MK-801. Our results provide direct in vivo electrophysiological evidence that ACC pyramidal cells undergo rapid and prolonged depolarization after digit amputation, and the amputation-induced depolarization in ACC neurons might be associated with the synaptic mechanisms for phantom pain.

Increase of delayed rectifier potassium currents in large aspiny neurons in the neostriatum following transient forebrain ischemia.

Large aspiny (LA) neurons in the neostriatum are resistant to cerebral ischemia whereas spiny neurons are highly vulnerable to the same insult. Excitotoxicity has been implicated as the major cause of neuronal damage after ischemia. Voltage-dependent potassium currents play important roles in controlling neuronal excitability and therefore influence the ischemic outcome. To reveal the ionic mechanisms underlying the ischemia-resistance, the delayed rectifier potassium currents (Ik) in LA neurons were studied before and at different intervals after transient forebrain ischemia using brain slices and acute dissociation preparations. The current density of Ik increased significantly 24 h after ischemia and returned to control levels 72 h following reperfusion. Among currents contributing to Ik, the margatoxin-sensitive currents increased 24 h after ischemia while the KCNQ/M current remained unchanged after ischemia. Activation of protein kinase A (PKA) down-regulated Ik in both control and ischemic LA neurons, whereas inhibition of PKA only up-regulated Ik and margatoxin-sensitive currents 72 h after ischemia, indicating an active PKA regulation on Ik at this time. Protein tyrosine kinases had a tonic inhibition on Ik to a similar extent before and after ischemia. Compared with that of control neurons, the spike width was significantly shortened 24 h after ischemia due to facilitated repolarization, which could be reversed by blocking margatoxin-sensitive currents. The increase of Ik in LA neurons might be one of the protective mechanisms against ischemic insult.

Asymmetrical changes of excitatory synaptic transmission in dopamine-denervated striatum after transient forebrain ischemia.

Spiny neurons in the neostriatum are highly vulnerable to cerebral ischemia. Recent studies have shown that the postischemic cell death in the right striatum was reduced after ipsilateral dopamine denervation whereas no protection was observed in the left striatum after dopamine denervation in the left side. In order to reveal the mechanisms of such asymmetrical protection, electrophysiological changes of dopamine-denervated striatal neurons were compared after ischemia between the left and right striatum using intracellular recording and staining techniques in vivo. No difference in cortically evoked initial excitatory postsynaptic potentials was found between the left and right striatum in intact animals after ipsilateral dopamine denervation. The initial excitatory postsynaptic potentials in the dopamine-denervated right striatum were suppressed after transient forebrain ischemia while no significant changes were found in the dopamine-denervated left striatum. Paired-pulse tests suggested that these changes involved presynaptic mechanisms. Although the incidence of a late depolarizing postsynaptic potential elicited by cortical stimulation increased after ischemia in both sides, the increase was greater in the left side. The analysis of current-voltage relationship of spiny neurons indicated that inward rectification in the left striatum transiently disappeared shortly after ischemia whereas that in the right side remained unchanged. The intrinsic excitability of spiny neurons in both sides were suppressed after ischemia, however, the suppression in the right side was stronger than in the left side. The above results demonstrate that after ipsilateral dopamine denervation, the depression of excitatory synaptic transmission and neuronal excitability in the right striatum is more severe than that in the left striatum following ischemia. The depression of excitatory synaptic transmission and neuronal excitability, therefore, might play an important role in neural protection after ischemic insult.

5-HT potentiation of the GABA(A) response in the rat sacral dorsal commissural neurones.

1. The modulatory effect of 5-hydroxytryptamine (5-HT) on the gamma-aminobutyric acid(A) (GABA(A)) response was investigated in the neurones freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin perforated patch recording configuration under the voltage-clamp conditions. 2. 5-HT potentiated GABA-induced Cl- current (IGABA) without affecting the reversal potential of IGABA and the apparent affinity of GABA to its receptor. 3. Alpha-Methyl-5-HT mimicked the potentiation effect of 5-HT on IGABA while ketanserine blocked it. 1-Oleoyl-2-acetyl-glycerol (OAG) potentiated IGABA, and the effect of 5-HT on IGABA was occluded by OAG pretreatment. In the presence of chelerythrine, 5-HT failed to potentiate IGABA, suggesting that protein kinase C (PKC) is involved in the pathway through which the activation of the 5-HT2 receptor potentiates the IGABA. 4. The facilitatory effect of 5-HT on IGABA remained in the presence of BAPTA-AM. LiCl also had no effect on 5-HT-induced potentiation of IGABA. 5. H-89, genistein, okadaic acid and pervanadate all had no effects on 5-HT potentiation of IGABA. Pertussis toxin treatment for 6-8 h did not block the facilitatory effect of 5-HT on IGABA. 6. The present results show that GABA(A) receptor in the rat SDCN could be modulated in situ by 5-HT, one of the major transmitters involved in the supraspinal control of nociception, and that the phosphorylation of GABA(A) receptor by PKC may be sufficient to support such modulation. The results also strongly support the hypothesis that the cotransmission by 5-HT and GABA has an important role in the spinal cord.

Taurine-activated chloride currents in the rat sacral dorsal commissural neurons.

The electrophysiological and pharmacological properties of taurine (Tau)-activated Cl- currents (ITau) were investigated in the dissociated rat sacral dorsal commissural nucleus (SDCN) neurons using the nystatin perforated patch recording configuration under voltage-clamp conditions. The reversal potential of ITau was close to the Cl- equilibrium potential. The ITau was not affected by a preceding GABA response but cross-desensitized by a preceding glycine (Gly) response. Strychnine (STR), picrotoxin (PIC), bicuculline (BIC) and Zn2+ suppressed the ITau in a concentration-dependent manner. The pharmacology of the ITau and Gly-induced response (IGly) was similar, though Zn2+ inhibition on ITau differed from that on IGly in being much slower in recovery. Serotonin potentiated the ITau via protein kinase C. The results indicate that both Tau and Gly act on a strychnine-sensitive site to open the same Cl- channels in the SDCN neurons, and suggest that Tau may act as a functional neurotransmitter in the mammalian SDCN.

Zn2+ depresses GABAA receptor mediated responses in acutely dissociated sacral dorsal commissural neurons.

The effects of Zn2+ on GABAA-receptor mediated responses in acutely isolated rat sacral dorsal commissural nucleus (SDCN) were studied using nystatin-perforated whole cell recording techniques. The results demonstrated that (1) GABA induced inward currents through activation of GABAA-receptor at a holding potential of -40 mV; (2) GABAA-receptor mediated responses were suppressed by Zn2+ in a reversible and voltage-independent manner; and (3) in the presence of Zn2+, the concentration-response curve of GABA-induced responses was shifted to the right in a parallel manner. The results suggest that Zn2+ allosterically depresses GABAA-receptor mediated currents.

[Enhancement of taurine-activated whole-cell currents by serotonin in rat sacral dorsal commissural neurons].

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of serotonin (5-hydroxytryptamine, 5-HT) on taurine (Tau)-activated current (ITau) was investigated in neurons dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results are as follows: (1) Tau acted on strychnine-sensitive glycine (Gly) receptors and elicited inward currents at a holding potential (VH) of -40 mV in SDCN neurons; (2)in a range of 0.01 to 100 mumol/L, 5-HT enhanced ITau in a concentration-dependent manner; (3) both the reversal potential of Tau and the binding affinity of Tau to Gly receptor were not affected by 5-HT; (4) when the neurons were loaded with 3 mumol/L chelerythrine, application of 1 mumol/L 5-HT failed to enhance ITau. The above results thus indicate that intracellular PKC pathway is at least partially involved in the enhancement of ITau by 5-HT. In accordance with our previous study, it is suggested that 5-HT may play an important antinociceptive function in SDCN through enhanced Gly and Tau inhibitory effects.

Motor neuron degeneration in a mouse model of seipinopathy.

Heterozygosity for missense mutations (N88S/S90L) in BSCL2 (Berardinelli-Seip congenital lipodystrophy type 2)/Seipin is associated with a broad spectrum of motoneuron diseases. To understand the underlying mechanisms how the mutations lead to motor neuropathy, we generated transgenic mice with neuron-specific expression of wild-type (tgWT) or N88S/S90L mutant (tgMT) human Seipin. Transgenes led to the broad expression of WT or mutant Seipin in the brain and spinal cord. TgMT, but not tgWT, mice exhibited late-onset altered locomotor activities and gait abnormalities that recapitulate symptoms of seipinopathy patients. We found loss of alpha motor neurons in tgMT spinal cord. Mild endoreticular stress was present in both tgMT and tgWT neurons; however, only tgMT mice exhibited protein aggregates and disrupted Golgi apparatus. Furthermore, autophagosomes were significantly increased, along with elevated light chain 3 (LC3)-II level in tgMT spinal cord, consistent with the activation of autophagy pathway in response to mutant Seipin expression and protein aggregation. These results suggest that induction of autophagy pathway is involved in the cellular response to mutant Seipin in seipinopathy and that motoneuron loss is a key pathogenic process underlying the development of locomotor abnormalities.

Transient enhancement of inhibitory synaptic transmission in hippocampal CA1 pyramidal neurons after cerebral ischemia.

Pyramidal neurons in hippocampal CA1 regions are highly sensitive to cerebral ischemia. Alterations of excitatory and inhibitory synaptic transmission may contribute to the ischemia-induced neuronal degeneration. However, little is known about the changes of GABAergic synaptic transmission in the hippocampus following reperfusion. We examined the GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons 12 and 24 h after transient forebrain ischemia in rats. The amplitudes of evoked inhibitory postsynaptic currents (eIPSCs) were increased significantly 12 h after ischemia and returned to control levels 24 h following reperfusion. The potentiation of eIPSCs was accompanied by an increase of miniature inhibitory postsynaptic current (mIPSC) amplitude, and an enhanced response to exogenous application of GABA, indicating the involvement of postsynaptic mechanisms. Furthermore, there was no obvious change of the paired-pulse ratio (PPR) of eIPSCs and the frequency of mIPSCs, suggesting that the potentiation of eIPSCs might not be due to the increased presynaptic release. Blockade of adenosine A1 receptors led to a decrease of eIPSCs amplitude in post-ischemic neurons but not in control neurons, without affecting the frequency of mIPSCs and the PPR of eIPSCs. Thus, tonic activation of adenosine A1 receptors might, at least in part, contribute to the enhancement of inhibitory synaptic transmission in CA1 neurons after forebrain ischemia. The transient enhancement of inhibitory neurotransmission might temporarily protect CA1 pyramidal neurons, and delay the process of neuronal death after cerebral ischemia.

Nicotine facilitates glycine release in the rat spinal dorsal horn.

1. Nicotinic effects on glycine release were investigated in slices of lumbar spinal cord using conventional whole-cell recordings. In most of the substantia gelatinosa (SG) neurons tested, nicotine increased the frequency of the glycinergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs). In a smaller proportion, nicotine evoked not only this same presynaptic response but also a postsynaptic response. 2. Nicotinic facilitation of glycinergic mIPSCs was investigated in mechanically dissociated SG neurons using nystatin-perforated patch recordings. Nicotine (3 x 10(-6) to 10(-5) M) reversibly enhanced the frequency of glycinergic mIPSCs without altering their amplitudes, thus indicating that nicotine facilitates glycine release through a presynaptic mechanism. 3. Choline, a selective alpha7 subunit of nicotinic acetylcholine receptor (nAChR) agonist, had no effect on the mIPSC frequency while anatoxin A, a broad-spectrum agonist of nAChR, facilitated the mIPSC frequency. 4. alpha-Bungarotoxin, a selective alpha7 subunit antagonist, failed to block the nicotinic facilitatory action. Mecamylamine, a broad-spectrum nicotinic antagonist, reversibly inhibited nicotinic action. Dihydro-beta-erythroidine, a selective antagonist of nAChRs containing alpha4-beta2 subunits, completely blocked nicotinic action. 5. Ca(2+)-free but not Cd(2+)-containing bath solutions blocked nicotinic actions. 6. We therefore conclude that nicotine facilitates glycine release in the substantia gelatinosa of the spinal dorsal horn via specific nAChRs containing alpha4-beta2 subunits. This action on a subset of presynaptic nAChRs may underlie nicotine's modulation of noxious signal transmission and provide a cellular mechanism for the analgesic function of nicotine.

Isolation of rat sacral dorsal commissural neurons.

UNLABELLED: To isolate rat sacral dorsal commissural neurons (SDCN). METHODS: Using enzymatic and mechanical dissociation techniques to isolate the neurons and using nystatin perforated patch technique to evaluate their functional state. RESULTS: The isolated neurons exhibited good responses to excitatory and inhibitory amino acids. The responses of SDCN to N-methyl-D-aspartate were markedly potentiated by substance P and trans-1-aminocyclopentane-1,3-dicarboxylate, whereas GABA responses were significantly potentiated by diazepam, pregnenolone, and pentobarbital. CONCLUSION: This preparation provides a satisfactory model for exploring the mechanisms of the SDCN in nociception and antinociception.

Dual effects of pentobarbital on rat sacral dorsal commissural neurons in vitro.

AIM: To study the effects of pentobarbital (PB) on acutely dissociated rat sacral dorsal commissural neurons (SDCN). METHODS: Nystatin-perforated patch clamp recording was used. RESULTS: (1) At a holding potential of -40 mV, PB induced inward Cl- current (IPB) in a concentration-dependent manner with a EC50 (95% confidence limits) of 416 (385-477) mumol.L-1 and a Hill coefficient of 1.08. (2) Picrotoxin reversibly blocked IPB. (3) The reversal potential of IPB was close to the Cl- equilibrium potential. (4) PB enhanced GABA-induced Cl- influx (IGABA). In the presence of PB 30 mumol.L-1, the EC50 (95% confidence limits) of IGABA decreased from 6.9 (5.4-8.4) mumol.L-1 to 3.5 (2.9-4.1) mumol.L-1. CONCLUSION: PB had dual effects on SDCN, facilitated GABAA receptor-mediated currents and at higher concentrations induced Cl- influx itself.