Cholinergic profiles in the Goettingen miniature pig (Sus scrofa domesticus) brain

Central cholinergic structures within the brain of the even‐toed hoofed Goettingen miniature domestic pig (Sus scrofa domesticus) were evaluated by immunohistochemical visualization of choline acetyltransferase (ChAT) and the low‐affinity neurotrophin receptor, p75^NTR. ChAT‐immunoreactive (‐ir) perikarya were seen in the olfactory tubercle, striatum, medial septal nucleus, vertical and horizontal limbs of the diagonal band of Broca, and the nucleus basalis of Meynert, medial habenular nucleus, zona incerta, neurosecretory arcuate nucleus, cranial motor nuclei III and IV, Edinger‐Westphal nucleus, parabigeminal nucleus, pedunculopontine nucleus, and laterodorsal tegmental nucleus. Cholinergic ChAT‐ir neurons were also found within transitional cortical areas (insular, cingulate, and piriform cortices) and hippocampus proper. ChAT‐ir fibers were seen throughout the dentate gyrus and hippocampus, in the mediodorsal, laterodorsal, anteroventral, and parateanial thalamic nuclei, the fasciculus retroflexus of Meynert, basolateral and basomedial amygdaloid nuclei, anterior pretectal and interpeduncular nuclei, as well as select laminae of the superior colliculus. Double immunofluorescence demonstrated that virtually all ChAT‐ir basal forebrain neurons were also p75^NTR‐positive. The present findings indicate that the central cholinergic system in the miniature pig is similar to other mammalian species. Therefore, the miniature pig may be an appropriate animal model for preclinical studies of neurodegenerative diseases where the cholinergic system is compromised. J. Comp. Neurol. 525:553–573, 2017. © 2016 Wiley Periodicals, Inc.     

Organization of alpha‐transducin immunoreactive system in the brain and retina of larval and young adult Sea Lamprey (Petromyzon marinus), and their relationship with other neural systems

We employed an anti‐transducin antibody (Gαt‐S), in combination with other markers, to characterize the Gαt‐S‐immunoreactive (ir) system in the CNS of the sea lamprey, Petromyzon marinus. Gαt‐S immunoreactivity was observed in some neuronal populations and numerous fibers distributed throughout the brain. Double Gαt‐S‐ and opsin‐ir neurons (putative photoreceptors) are distributed in the hypothalamus (postoptic commissure nucleus, dorsal and ventral hypothalamus) and caudal diencephalon, confirming results of García‐Fernández et al. (Cell and Tissue Research, 288, 267–278, 1997). Singly Gαt‐S‐ir cells were observed in the midbrain and hindbrain, increasing the known populations. Our results reveal for the first time in vertebrates the extensive innervation of many brain regions and the spinal cord by Gαt‐S‐ir fibers. The Gαt‐S innervation of the habenula is very selective, fibers densely innervating the lamprey homologue of the mammalian medial nucleus (Stephenson‐Jones et al., Proceedings of the National Academy of Sciences of the United States of America, 109, E164–E173, 2012), but not the lateral nucleus homologue. The lamprey neurohypophysis was not innervated by Gαt‐S‐ir fibers. We also analyzed by double immunofluorescence the relation of this system with other systems. A dopaminergic marker (TH), serotonin (5‐HT) or GABA do not co‐localize with Gαt‐S‐ir neurons although codistribution of fibers was observed. Codistribution of Gαt‐S‐ir fibers and isolectin‐labeled extrabulbar primary olfactory fibers was observed in the striatum and hypothalamus. Neurobiotin retrograde transport from the spinal cord combined with immunofluorescence revealed spinal‐projecting Gαt‐S‐ir reticular neurons in the caudal hindbrain. Present results in an ancient vertebrate reveal for the first time a collection of brain targets of Gαt‐S‐ir neurons, suggesting they might mediate non‐visual modulation by light in many systems.     

Comparative analysis of monoaminergic cerebrospinal fluid‐contacting cells in Osteichthyes (bony vertebrates)

Cerebrospinal fluid‐contacting (CSF‐c) cells containing monoamines such as dopamine (DA) and serotonin (5‐HT) occur in the periventricular zones of the hypothalamic region of most vertebrates except for placental mammals. Here we compare the organization of the CSF‐c cells in chicken, Xenopus, and zebrafish, by analyzing the expression of synthetic enzymes of DA and 5‐HT, respectively, tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), and draw an evolutionary scenario for this cell population. Due to the lack of TH immunoreactivity in this region, the hypothalamic CSF‐c cells have been thought to take up DA from the ventricle instead of synthesizing it. We demonstrate that a second TH gene (TH2) is expressed in the CSF‐c cells of all the three species, suggesting that these cells do indeed synthetize DA. Furthermore, we found that many CSF‐c cells coexpress TH2 and TPH1 and contain both DA and 5‐HT, a dual neurotransmitter phenotype hitherto undescribed in the brain of any vertebrate. The similarities of CSF‐c cells in chicken, Xenopus, and zebrafish suggest that these characteristics are inherited from the common ancestor of the Osteichthyes. A significant difference between tetrapods and teleosts is that teleosts possess an additional CSF‐c cell population around the posterior recess (PR) that has emerged in specific groups of Actinopterygii. Our comparative analysis reveals that the hypothalamus in mammals and teleosts has evolved in a divergent manner: placental mammals have lost the monoaminergic CSF‐c cells, while teleosts have increased their relative number.     

Somatic and neuritic spines on tyrosine hydroxylase–immunopositive cells of rat retina

Dopamine‐ and tyrosine hydroxylase–immunopositive cells (TH cells) modulate visually driven signals as they flow through retinal photoreceptor, bipolar, and ganglion cells. Previous studies suggested that TH cells release dopamine from varicose axons arborizing in the inner and outer plexiform layers after glutamatergic synapses depolarize TH cell dendrites in the inner plexiform layer and these depolarizations propagate to the varicosities. Although it has been proposed that these excitatory synapses are formed onto appendages resembling dendritic spines, spines have not been found on TH cells of most species examined to date or on TH cell somata that release dopamine when exposed to glutamate receptor agonists. By use of protocols that preserve proximal retinal neuron morphology, we have examined the shape, distribution, and synapse‐related immunoreactivity of adult rat TH cells. We report here that TH cell somata, tapering and varicose inner plexiform layer neurites, and varicose outer plexiform layer neurites all bear spines, that some of these spines are immunopositive for glutamate receptor and postsynaptic density proteins (viz., GluR1, GluR4, NR1, PSD‐95, and PSD‐93), that TH cell somata and tapering neurites are also immunopositive for a γ‐aminobutyric acid (GABA) receptor subunit (GABA_AR_α1), and that a synaptic ribbon‐specific protein (RIBEYE) is found adjacent to some colocalizations of GluR1 and TH in the inner plexiform layer. These results identify previously undescribed sites at which glutamatergic and GABAergic inputs may stimulate and inhibit dopamine release, especially at somata and along varicose neurites that emerge from these somata and arborize in various levels of the retina. J. Comp. Neurol. 525:1707–1730, 2017. © 2016 Wiley Periodicals, Inc.     

Decreased approach behavior and nucleus accumbens immediate early gene expression in response to Parkinsonian ultrasonic vocalizations in rats

Many individuals with Parkinson disease (PD) have difficulty producing normal speech and voice, resulting in problems with interpersonal communication and reduced quality of life. Translational animal models of communicative dysfunction have been developed to assess disease pathology. However, it is unknown whether acoustic feature changes associated with vocal production deficits in these animal models lead to compromised communication. In rodents, male ultrasonic vocalizations (USVs) have a well-established role in functional inter-sexual communication. To test whether acoustic deficits in USVs observed in a PTEN-induced putative kinase 1 (PINK1) knockout (KO) PD rat model compromise communication, we presented recordings of male PINK1 KO USVs and normal wild-type (WT) USVs to female rat listeners. We measured approached behavior and immediate early gene expression (c-Fos) in brain regions implicated in auditory processing and sexual motivation. Our results suggest that females show reduced approach in response to PINK1 KO USVs compared with WT. Moreover, females exposed to PINK1 KO USVs had lower c-Fos immunolabeling in the nucleus accumbens, a region implicated in sexual motivation. These results are the first to demonstrate that vocalization deficits in a rat PD model result in compromised communication. Thus, the PINK1 KO PD model may be valuable for assessing treatments aimed at restoring vocal communicative function.     

Functions of corazonin and histamine in light entrainment of the circadian pacemaker in the Madeira cockroach,Rhyparobia maderae

The circadian pacemaker of the Madeira cockroach, Rhyparobia (Leucophaea) maderae, is located in the accessory medulla (AME). Ipsi‐ and contralateral histaminergic compound eyes are required for photic entrainment. Light pulses delay locomotor activity rhythm during the early night and advance it during the late night. Thus, different neuronal pathways might relay either light‐dependent delays or advances to the clock. Injections of neuroactive substances combined with running‐wheel assays suggested that GABA, pigment‐dispersing factor, myoinhibitory peptides (MIPs), and orcokinins (ORCs) were part of both entrainment pathways, whereas allatotropin (AT) only delayed locomotor rhythms at the early night. To characterize photic entrainment further, histamine and corazonin were injected. Histamine injections resulted in light‐like phase delays and advances, indicating that the neurotransmitter of the compound eyes participates in both entrainment pathways. Because injections of corazonin only advanced during the late subjective night, it was hypothesized that corazonin is only part of the advance pathway. Multiple‐label immunocytochemistry in combination with neurobiotin backfills demonstrated that a single cell expressed corazonin in the optic lobes that belonged to the group of medial AME interneurons. It colocalized GABA and MIP but not AT or ORC immunoreactivity. Corazonin‐immunoreactive (‐ir) terminals overlapped with projections of putatively light‐sensitive interneurons from the ipsi‐ and contralateral compound eye. Thus, we hypothesize that the corazonin‐ir medial neuron integrates ipsi‐ and contralateral light information as part of the phase‐advancing light entrainment pathway to the circadian clock. J. Comp. Neurol. 525:1250–1272, 2017. © 2016 Wiley Periodicals, Inc.     

Brain region‐dependent differential expression of alpha‐synuclein

α‐Synuclein, the major constituent of Lewy bodies (LBs), is normally expressed in presynapses and is involved in synaptic function. Abnormal intracellular aggregation of α‐synuclein is observed as LBs and Lewy neurites in neurodegenerative disorders, such as Parkinson's disease (PD) or dementia with Lewy bodies. Accumulated evidence suggests that abundant intracellular expression of α‐synuclein is one of the risk factors for pathological aggregation. Recently, we reported differential expression patterns of α‐synuclein between excitatory and inhibitory hippocampal neurons. Here we further investigated the precise expression profile in the adult mouse brain with special reference to vulnerable regions along the progression of idiopathic PD. The results show that α‐synuclein was highly expressed in the neuronal cell bodies of some early PD‐affected brain regions, such as the olfactory bulb, dorsal motor nucleus of the vagus, and substantia nigra pars compacta. Synaptic expression of α‐synuclein was mostly accompanied by expression of vesicular glutamate transporter‐1, an excitatory presynaptic marker. In contrast, expression of α‐synuclein in the GABAergic inhibitory synapses was different among brain regions. α‐Synuclein was clearly expressed in inhibitory synapses in the external plexiform layer of the olfactory bulb, globus pallidus, and substantia nigra pars reticulata, but not in the cerebral cortex, subthalamic nucleus, or thalamus. These results suggest that some neurons in early PD‐affected human brain regions express high levels of perikaryal α‐synuclein, as happens in the mouse brain. Additionally, synaptic profiles expressing α‐synuclein are different in various brain regions. J. Comp. Neurol. 524:1236–1258, 2016. © 2015 Wiley Periodicals, Inc.     

Normal development of spinal axons in early embryo stages and posterior locomotor function is independent of GAL‐1

It was recently described that Galectin‐1 (Gal‐1) promotes axonal growth after spinal cord injury. This effect depends on protein dimerization, since monomeric Gal‐1 fails to stimulate axonal re‐growth. Gal‐1 is expressed in vivo at concentrations that favor the monomeric species. The aim of the present study is to investigate whether endogenous Gal‐1 is required for spinal axon development and normal locomotor behavior in mice. In order to characterize axonal development, we used a novel combination of 3‐DISCO technique with 1‐photon microscopy and epifluorescence microscopy under high power LED illumination, followed by serial image section deconvolution and 3‐D reconstruction. Cleared whole lgals‐1^‐/‐ embryos were used to analyze the 3‐D cytoarchitecture of motor, commissural, and sensory axons. This approach allowed us to evaluate axonal development, including the number of fibers, fluorescence density of the fiber tracts, fiber length as well as the morphology of axonal sprouting, deep within the tissue. Gal‐1 deficient embryos did not show morphological/anatomical alterations in any of the axonal populations and parameters analyzed. In addition, specific guidance receptor PlexinA4 did not change its axonal localization in the absence of Gal‐1. Finally, Gal‐1 deficiency did not change normal locomotor activity in post‐natal animals. Taken together, our results show that development of spinal axons as well as the locomotor abilities observed in adult mice are independent of Gal‐1. Supporting our previous observations, the present study further validates the use of lgals‐1^‐/‐ mice to develop spinal cord‐ or traumatic brain injury models for the evaluation of the regenerative action of Gal‐1.     

Differential susceptibility of cortical and subcortical inhibitory neurons and astrocytes in the long term following diffuse traumatic brain injury

Long‐term diffuse traumatic brain injury (dTBI) causes neuronal hyperexcitation in supragranular layers in sensory cortex, likely through reduced inhibition. Other forms of TBI affect inhibitory interneurons in subcortical areas but it is unknown if this occurs in cortex, or in any brain area in dTBI. We investigated dTBI effects on inhibitory neurons and astrocytes in somatosensory and motor cortex, and hippocampus, 8 weeks post‐TBI. Brains were labeled with antibodies against calbindin (CB), parvalbumin (PV), calretinin (CR) and neuropeptide Y (NPY), and somatostatin (SOM) and glial fibrillary acidic protein (GFAP), a marker for astrogliosis during neurodegeneration. Despite persistent behavioral deficits in rotarod performance up to the time of brain extraction (TBI = 73.13 ± 5.23% mean ± SEM, Sham = 92.29 ± 5.56%, P < 0.01), motor cortex showed only a significant increase, in NPY neurons in supragranular layers (mean cells/mm² ± SEM, Sham = 16 ± 0.971, TBI = 25 ± 1.51, P = 0.001). In somatosensory cortex, only CR⁺ neurons showed changes, being decreased in supragranular (TBI = 19 ± 1.18, Sham = 25 ± 1.10, P < 0.01) and increased in infragranular (TBI = 28 ± 1.35, Sham = 24 ± 1.07, P < 0.05) layers. Heterogeneous changes were seen in hippocampal staining: CB⁺ decreased in dentate gyrus (TBI = 2 ± 0.382, Sham = 4 ± 0.383, P < 0.01), PV⁺ increased in CA1 (TBI = 39 ± 1.26, Sham = 33 ± 1.69, P < 0.05) and CA2/3 (TBI = 26 ± 2.10, Sham = 20 ± 1.49, P < 0.05), and CR⁺ decreased in CA1 (TBI = 10 ± 1.02, Sham = 14 ± 1.14, P < 0.05). Astrogliosis significantly increased in corpus callosum (TBI = 6.7 ± 0.69, Sham = 2.5 ± 0.38; P = 0.007). While dTBI effects on inhibitory neurons appear region‐ and type‐specific, a common feature in all cases of decrease was that changes occurred in dendrite targeting interneurons involved in neuronal integration. J. Comp. Neurol. 524:3530–3560, 2016. © 2016 Wiley Periodicals, Inc.     

Basal brain oxidative and nitrative stress levels are finely regulated by the interplay between superoxide dismutase 2 and p53

Superoxide dismutases (SODs) are the primary reactive oxygen species (ROS)‐scavenging enzymes of the cell and catalyze the dismutation of superoxide radicals O₂^– to H₂O₂ and molecular oxygen (O₂). Among the three forms of SOD identified, manganese‐containing SOD (MnSOD, SOD2) is a homotetramer located wholly in the mitochondrial matrix. Because of the SOD2 strategic location, it represents the first mechanism of defense against the augmentation of ROS/reactive nitrogen species levels in the mitochondria for preventing further damage. This study seeks to understand the effects that the partial lack (SOD2^?/+) or the overexpression (TgSOD2) of MnSOD produces on oxidative/nitrative stress basal levels in different brain isolated cellular fractions (i.e., mitochondrial, nuclear, cytosolic) as well as in the whole‐brain homogenate. Furthermore, because of the known interaction between SOD2 and p53 protein, this study seeks to clarify the impact that the double mutation has on oxidative/nitrative stress levels in the brain of mice carrying the double mutation (p53^?/? × SOD2^?/+ and p53^?/? × TgSOD2). We show that each mutation affects mitochondrial, nuclear, and cytosolic oxidative/nitrative stress basal levels differently, but, overall, no change or reduction of oxidative/nitrative stress levels was found in the whole‐brain homogenate. The analysis of well‐known antioxidant systems such as thioredoxin‐1 and Nrf2/HO‐1/BVR‐A suggests their potential role in the maintenance of the cellular redox homeostasis in the presence of changes of SOD2 and/or p53 protein levels. © 2015 Wiley Periodicals, Inc.     

Coexpression of auxiliary subunits KChIP and DPPL in potassium channel Kv4‐positive nociceptors and pain‐modulating spinal interneurons

Subthreshold A‐type K⁺ currents (I_SAs) have been recorded from the somata of nociceptors and spinal lamina II excitatory interneurons, which sense and modulate pain, respectively. Kv4 channels are responsible for the somatodendritic I_SAs. Accumulative evidence suggests that neuronal Kv4 channels are ternary complexes including pore‐forming Kv4 subunits and two types of auxiliary subunits: K⁺ channel‐interacting proteins (KChIPs) and dipeptidyl peptidase‐like proteins (DPPLs). Previous reports have shown Kv4.3 in a subset of nonpeptidergic nociceptors and Kv4.2/Kv4.3 in certain spinal lamina II excitatory interneurons. However, whether and which KChIP and DPPL are coexpressed with Kv4 in these I_SA‐expressing pain‐related neurons is unknown. In this study we mapped the protein distribution of KChIP1, KChIP2, KChIP3, DPP6, and DPP10 in adult rat dorsal root ganglion (DRG) and spinal cord by immunohistochemistry. In the DRG, we found colocalization of KChIP1, KChIP2, and DPP10 in the somatic surface and cytoplasm of Kv4.3(+) nociceptors. KChIP3 appears in most Aβ and Aδ sensory neurons as well as a small population of peptidergic nociceptors, whereas DPP6 is absent in sensory neurons. In the spinal cord, KChIP1 is coexpressed with Kv4.3 in the cell bodies of a subset of lamina II excitatory interneurons, while KChIP1, KChIP2, and DPP6 are colocalized with Kv4.2 and Kv4.3 in their dendrites. Within the dorsal horn, besides KChIP3 in the inner lamina II and lamina III, we detected DPP10 in most projection neurons, which transmit pain signal to brain. The results suggest the existence of Kv4/KChIP/DPPL ternary complexes in I_SA‐expressing nociceptors and pain‐modulating spinal interneurons. J. Comp. Neurol. 524:846–873, 2016. © 2015 Wiley Periodicals, Inc.     

Sphingolipids regulate neuromuscular synapse structure and function in Drosophila

Sphingolipids are found in abundance at synapses and have been implicated in regulation of synapse structure, function, and degeneration. Their precise role in these processes, however, remains obscure. Serine Palmitoyl‐transferase (SPT) is the first enzymatic step for synthesis of sphingolipids. Analysis of the Drosophila larval neuromuscular junction (NMJ) revealed mutations in the SPT enzyme subunit, lace/SPTLC2 resulted in deficits in synaptic structure and function. Although NMJ length is normal in lace mutants, the number of boutons per NMJ is reduced to ～50% of the wild type number. Synaptic boutons in lace mutants are much larger but show little perturbation to the general ultrastructure. Electrophysiological analysis of lace mutant synapses revealed strong synaptic transmission coupled with predominance of depression over facilitation. The structural and functional phenotypes of lace mirrored aspects of Basigin (Bsg), a small Ig‐domain adhesion molecule also known to regulate synaptic structure and function. Mutant combinations of lace and Bsg generated large synaptic boutons, while lace mutants showed abnormal accumulation of Bsg at synapses, suggesting that Bsg requires sphingolipid to regulate structure of the synapse. In support of this, we found Bsg to be enriched in lipid rafts. Our data points to a role for sphingolipids in the regulation and fine‐tuning of synaptic structure and function while sphingolipid regulation of synaptic structure may be mediated via the activity of Bsg.     

Greater neurodegeneration and behavioral deficits after single closed head traumatic brain injury in adolescent versus adult male mice

Traumatic brain injury (TBI) is a major public health concern affecting 2.8 million people per year in the United States, of whom about 1 million are children under 19 years old. Animal models of TBI have been developed and used in multiple ages of animals, but direct comparisons of adult and adolescent populations are rare. The current studies were undertaken to directly compare outcomes between adult and adolescent male mice, using a closed head, single-impact model of TBI. Six-week-old adolescent and 9-week-old adult male mice were subjected to mild–moderate TBI. Histological measures for neurodegeneration, gliosis, and microglial neuroinflammation, and behavioral tests of locomotion and memory were performed. Adolescent TBI mice have increased mortality (Χ² = 20.72, p < 0.001) compared to adults. There is also evidence of hippocampal neurodegeneration in adolescents that is not present in adults. Hippocampal neurodegeneration correlates with histologic activation of microglia, but not with increased astrogliosis. Adults and adolescents have similar locomotion deficits after TBI that recover by 16 days postinjury. Adolescents have memory deficits as evidenced by impaired novel object recognition between 3–4 and 4–16 days postinjury (F_1,26 = 5.23, p = 0.031) while adults do not. In conclusion, adults and adolescents within a close age range (6–9 weeks) respond to TBI differently. Adolescents are more severely affected by mortality, neurodegeneration, and inflammation in the hippocampus compared to adults. Adolescents, but not adults, have worse memory performance after TBI that lasts at least 16 days postinjury.     

Dysfunctional RNA-binding protein biology and neurodegeneration in experimental autoimmune encephalomyelitis in female mice

Altered stress granule (SG) and RNA-binding protein (RBP) biology have been shown to contribute to the pathogenesis of several neurodegenerative diseases, yet little is known about their role in multiple sclerosis (MS). Pathological features associated with dysfunctional RBPs include RBP mislocalization from its normal nuclear location to the cytoplasm and the formation of chronic SGs. We tested the hypothesis that altered SG and RBP biology might contribute to the neurodegeneration in experimental autoimmune encephalomyelitis (EAE). C57BL/6 female mice were actively immunized with MOG_35-55 to induce EAE. Spinal cords were examined for mislocalization of the RBPs, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and TAR-DNA binding protein-43 (TDP-43), SGs, neurodegeneration (SMI-32), T cells (CD3), and macrophages (CD68). In contrast to naive mice, mice with EAE showed SG formation (p < 0.0001) and mislocalization of hnRNP A1 (p < 0.05) in neurons of the ventral spinal cord gray matter, which correlated with clinical score (R = 0.8104, p = 0.0253). In these same areas, there was a neuronal loss (p < 0.0001) and increased SMI-32 immunoreactivity (both markers of neurodegeneration) and increased staining for CD3⁺ T cells and IFN-gamma. These findings recapitulate the SG and RBP biology and markers of neurodegeneration in MS tissues and suggest that altered SG and RBP biology contribute to the neurodegeneration in EAE, which might also apply to the pathogenesis of MS.     

The impact of parkinson's disease on the cortical mechanisms that support auditory–motor integration for voice control

Several studies have shown sensorimotor deficits in speech processing in individuals with idiopathic Parkinson's disease (PD). The underlying neural mechanisms, however, remain poorly understood. In the present event‐related potential (ERP) study, 18 individuals with PD and 18 healthy controls were exposed to frequency‐altered feedback (FAF) while producing a sustained vowel and listening to the playback of their own voice. Behavioral results revealed that individuals with PD produced significantly larger vocal compensation for pitch feedback errors than healthy controls, and exhibited a significant positive correlation between the magnitude of their vocal responses and the variability of their unaltered vocal pitch. At the cortical level, larger P2 responses were observed for individuals with PD compared with healthy controls during active vocalization due to left‐lateralized enhanced activity in the superior and inferior frontal gyrus, premotor cortex, inferior parietal lobule, and superior temporal gyrus. These two groups did not differ, however, when they passively listened to the playback of their own voice. Individuals with PD also exhibited larger P2 responses during active vocalization when compared with passive listening due to enhanced activity in the inferior frontal gyrus, precental gyrus, postcentral gyrus, and middle temporal gyrus. This enhancement effect, however, was not observed for healthy controls. These findings provide neural evidence for the abnormal auditory–vocal integration for voice control in individuals with PD, which may be caused by their deficits in the detection and correction of errors in voice auditory feedback. Hum Brain Mapp 37:4248–4261, 2016. © 2016 Wiley Periodicals, Inc.     

GABAA and GABAB receptor subunit localization on neurochemically identified neurons of the human subthalamic nucleus

The subthalamic nucleus (STN) is a critical excitatory signaling center within the basal ganglia circuitry. The activity of subthalamic neurons is tightly controlled by upstream inhibitory signaling centers in the basal ganglia. In this study, we used immunohistochemical techniques to firstly, visualize and quantify the STN neurochemical organization based on neuronal markers including parvalbumin (PV), calretinin (CR), SMI‐32, and GAD_65/67. Secondly, we characterized the detailed regional, cellular and subcellular expression of GABA_A (α₁, α₂, α₃, β_2/3, and γ₂) and GABA_B (R1 and R2) receptor subunits within the normal human STN. Overall, we found seven neurochemically distinct populations of principal neurons in the human STN. The three main populations detected were: (a) triple‐labeled PV⁺/CR⁺/SMI32⁺; (b) double‐labeled PV⁺/CR⁺; and (c) single‐labeled CR⁺ neurons. Subthalamic principal neurons were found to express GABA_A receptor subunits α₁, α₃, β_2/3, γ₂, and GABA_B receptor subunits R1 and R2. However, no expression of GABA_A receptor α₂ subunit was detected. We also found a trend of increasing regional staining intensity for all positive GABA_A receptor subunits from the dorsolateral pole to ventromedial extremities. The GAD⁺ interneurons showed relatively low expression of GABA_A receptor subunits. These results provide the morphological basis of GABAergic transmission within the normal human subthalamic nucleus and evidence of GABA innervation through both GABA_A and GABA_B receptors on subthalamic principal neurons.     

Localization of the M2 muscarinic cholinergic receptor in dendrites,cholinergic terminals,and noncholinergic terminals in the rat basolateral amygdala: An ultrastructural analysis

Activation of M2 muscarinic receptors (M2Rs) in the rat anterior basolateral nucleus (BLa) is critical for the consolidation of memories of emotionally arousing events. The present investigation used immunocytochemistry at the electron microscopic level to determine which structures in the BLa express M2Rs. In addition, dual localization of M2R and the vesicular acetylcholine transporter protein (VAChT), a marker for cholinergic axons, was performed to determine whether M2R is an autoreceptor in cholinergic axons innervating the BLa. M2R immunoreactivity (M2R‐ir) was absent from the perikarya of pyramidal neurons, with the exception of the Golgi complex, but was dense in the proximal dendrites and axon initial segments emanating from these neurons. Most perikarya of nonpyramidal neurons were also M2R–negative. About 95% of dendritic shafts and 60% of dendritic spines were M2 immunoreactive (M2R⁺). Some M2R⁺ dendrites had spines, suggesting that they belonged to pyramidal cells, whereas others had morphological features typical of nonpyramidal neurons. M2R‐ir was also seen in axon terminals, most of which formed asymmetrical synapses. The main targets of M2R⁺ terminals forming asymmetrical (putative excitatory) synapses were dendritic spines, most of which were M2R⁺. The main targets of M2R⁺ terminals forming symmetrical (putative inhibitory or neuromodulatory) synapses were unlabeled perikarya and M2R⁺ dendritic shafts. M2R‐ir was also seen in VAChT⁺ cholinergic terminals, indicating a possible autoreceptor role. These findings suggest that M2R‐mediated mechanisms in the BLa are very complex, involving postsynaptic effects in dendrites as well as regulating release of glutamate, γ‐aminobutyric acid, and acetylcholine from presynaptic axon terminals. J. Comp. Neurol. 524:2400–2417, 2016. © 2016 Wiley Periodicals, Inc.     

Topographic organization and possible function of the posterior optic tubercles in the brain of the desert locust Schistocerca gregaria

Migrating desert locusts, Schistocerca gregaria, are able to use the skylight polarization pattern for navigation. They detect polarized light with a specialized dorsal rim area in their compound eye. After multistage processing, polarization signals are transferred to the central complex, a midline‐spanning brain area involved in locomotor control. Polarization‐sensitive tangential neurons (TB‐neurons) of the protocerebral bridge, a part of the central complex, give rise to a topographic arrangement of preferred polarization angles in the bridge, suggesting that the central complex acts as an internal sky compass. TB‐neurons connect the protocerebral bridge with two adjacent brain areas, the posterior optic tubercles. To analyze the polarotopic organization of the central complex further, we investigated the number and morphologies of TB‐neurons and the presence and colocalization of three neuroactive substances in these neurons. Triple immunostaining with antisera against Diploptera punctata allatostatin (Dip‐AST), Manduca sexta allatotropin (Mas‐AT), and serotonin (5HT) raised in the same host species revealed three spatially distinct TB‐neuron clusters, each consisting of 10 neurons per hemisphere: cluster 1 and 3 showed Dip‐AST/5HT immunostaining, whereas cluster 2 showed Dip‐AST/Mas‐AT immunostaining. Five subtypes of TB‐neuron could be distinguished based on ramification patterns. Corresponding to ramification domains in the protocerebral bridge, the neurons invaded distinct but overlapping layers within the posterior optic tubercle. Similarly, neurons interconnecting the tubercles of the two hemispheres also targeted distinct layers of these neuropils. From these data we propose a neuronal circuit that may be suited to stabilize the internal sky compass in the central complex of the locust. J. Comp. Neurol. 523:1589–1607, 2015. © 2015 Wiley Periodicals, Inc.     

Synapsin knockdown is associated with decreased neurite outgrowth,functional synaptogenesis impairment,and fast high‐frequency neurotransmitter release

Synapsins (Syns) are an evolutionarily conserved family of synaptic vesicle‐associated proteins related to fine tuning of synaptic transmission. Studies with mammals have partially clarified the different roles of Syns; however, the presence of different genes and isoforms and the development of compensatory mechanisms hinder accurate data interpretation. Here, we use a simple in vitro monosynaptic Helix neuron connection, reproducing an in vivo physiological connection as a reliable experimental model to investigate the effects of Syn knockdown. Cells overexpressing an antisense construct against Helix Syn showed a time‐dependent decrease of Syn immunostaining, confirming protein loss. At the morphological level, Syn‐silenced cells showed a reduction in neurite linear outgrowth and branching and in the size and number of synaptic varicosities. Functionally, Syn‐silenced cells presented a reduced ability to form synaptic connections; however, functional chemical synapses showed similar basal excitatory postsynaptic potentials and similar short‐term plasticity paradigms. In addition, Syn‐silenced cells presented faster neurotransmitter release and decreased postsynaptic response toward the end of long tetanic presynaptic stimulations, probably related to an impairment of the synaptic vesicle trafficking resulting from a different vesicle handling, with an increased readily releasable pool and a compromised reserve pool. © 2015 Wiley Periodicals, Inc.