Three-Dimensional Spatial Analyses of Cholinergic Neuronal Distributions Across The Mouse Septum, Nucleus Basalis, Globus Pallidus, Nucleus Accumbens, and Caudate-Putamen.

Neuronal networks are regulated by three-dimensional spatial and structural properties. Despite robust evidence of functional implications in the modulation of cognition, little is known about the three-dimensional internal organization of cholinergic networks in the forebrain. Cholinergic networks in the forebrain primarily occur in subcortical nuclei, specifically the septum, nucleus basalis, globus pallidus, nucleus accumbens, and the caudate-putamen. Therefore, the present investigation analyzed the three-dimensional spatial organization of 14,000 cholinergic neurons that expressed choline acetyltransferase (ChAT) in these subcortical nuclei of the mouse forebrain. Point process theory and graph signal processing techniques identified three topological principles of organization. First, cholinergic interneuronal distance is not uniform across brain regions. Specifically, in the septum, globus pallidus, nucleus accumbens, and the caudate-putamen, the cholinergic neurons were clustered compared with a uniform random distribution. In contrast, in the nucleus basalis, the cholinergic neurons had a spatial distribution of greater regularity than a uniform random distribution. Second, a quarter of the caudate-putamen is composed of axonal bundles, yet the spatial distribution of cholinergic neurons remained clustered when axonal bundles were accounted for. However, comparison with an inhomogeneous Poisson distribution showed that the nucleus basalis and caudate-putamen findings could be explained by density gradients in those structures. Third, the number of cholinergic neurons varies as a function of the volume of a specific brain region but cell body volume is constant across regions. The results of the present investigation provide topographic descriptions of cholinergic somata distribution and axonal conduits, and demonstrate spatial differences in cognitive control networks. The study provides a comprehensive digital database of the total population of ChAT-positive neurons in the reported structures, with the x,y,z coordinates of each neuron at micrometer resolution. This information is important for future digital cellular atlases and computational models of the forebrain cholinergic system enabling models based on actual spatial geometry.

Forebrain projection neurons target functionally diverse respiratory control areas in the midbrain, pons, and medulla oblongata.

Eupnea is generated by neural circuits located in the ponto-medullary brainstem, but can be modulated by higher brain inputs which contribute to volitional control of breathing and the expression of orofacial behaviors, such as vocalization, sniffing, coughing, and swallowing. Surprisingly, the anatomical organization of descending inputs that connect the forebrain with the brainstem respiratory network remains poorly defined. We hypothesized that descending forebrain projections target multiple distributed respiratory control nuclei across the neuroaxis. To test our hypothesis, we made discrete unilateral microinjections of the retrograde tracer cholera toxin subunit B in the midbrain periaqueductal gray (PAG), the pontine Kölliker-Fuse nucleus (KFn), the medullary Bötzinger complex (BötC), pre-BötC, or caudal midline raphé nuclei. We quantified the regional distribution of retrogradely labeled neurons in the forebrain 12-14 days postinjection. Overall, our data reveal that descending inputs from cortical areas predominantly target the PAG and KFn. Differential forebrain regions innervating the PAG (prefrontal, cingulate cortices, and lateral septum) and KFn (rhinal, piriform, and somatosensory cortices) imply that volitional motor commands for vocalization are specifically relayed via the PAG, while the KFn may receive commands to coordinate breathing with other orofacial behaviors (e.g., sniffing, swallowing). Additionally, we observed that the limbic or autonomic (interoceptive) systems are connected to broadly distributed downstream bulbar respiratory networks. Collectively, these data provide a neural substrate to explain how volitional, state-dependent, and emotional modulation of breathing is regulated by the forebrain.

Cell-Type-Specific Metabolic Profiling Achieved by Combining Desorption Electrospray Ionization Mass Spectrometry Imaging and Immunofluorescence Staining.

Cell-type-specific metabolic profiling in tissue with heterogeneous composition has been of great interest across all mass spectrometry imaging (MSI) technologies. We report here a powerful new chemical imaging capability in desorption electrospray ionization (DESI) MSI, which enables cell-type-specific and in situ metabolic profiling in complex tissue samples. We accomplish this by combining DESI-MSI with immunofluorescence staining using specific cell-type markers. We take advantage of the variable frequency of each distinct cell type in the lateral septal nucleus (LSN) region of mouse forebrain. This allows computational deconvolution of the cell-type-specific metabolic profile in neurons and astrocytes by convex optimization-a machine learning method. Based on our approach, we observed 107 metabolites that show different distributions and intensities between astrocytes and neurons. We subsequently identified 23 metabolites using high-resolution mass spectrometry (MS) and tandem MS, which include small metabolites such as adenosine and N-acetylaspartate previously associated with astrocytes and neurons, respectively, as well as accumulation of several phospholipid species in neurons which have not been studied before. Overall, this method overcomes the relatively low spatial resolution of DESI-MSI and provides a new platform for in situ metabolic investigation at the cell-type level in complex tissue samples with heterogeneous cell-type composition.

SNRIs achieve faster antidepressant effects than SSRIs by elevating the concentrations of dopamine in the forebrain.

Major depressive disorder (MDD) is a severe mental disorder with a high disability rate worldwide. Selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs) are the most common agents for antidepressant use. SSRIs and SNRIs are believed to achieve antidepressant effects through the activation of serotonergic or noradrenergic systems. However, whether the dopaminergic system is involved remains unclear. In our study, a genetically encoded dopamine sensor and in vivo fiber photometry recordings were used to measure the dopamine concentrations in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) after acute intraperitoneal injection of SSRIs or SNRIs. Combined with the behavioral tests, we found that SNRIs increased dopamine concentrations in both the mPFC and the NAc and showed faster antidepressant effects than SSRIs. To verify the enhanced dopamine levels induce the faster antidepressant effects of SNRIs, we employed dopamine receptor antagonists to specifically block the dopaminergic function. The results showed that the faster antidepressant effects of SNRIs were weakened by the dopamine receptor antagonists. Altogether, our study reveals that SNRIs achieve faster antidepressant effects than SSRIs by elevating the dopamine concentrations in the mPFC and the NAc. Our work proposes further mechanisms for the first-line antidepressants, which provides more basis for clinical treatments. This article is part of the special issue on Stress, Addiction and Plasticity.

Immunohistochemical distribution of 10 GABAA receptor subunits in the forebrain of the rhesus monkey Macaca mulatta.

GABAA receptors are composed of five subunits arranged around a central chloride channel. Their subunits originate from different genes or gene families. The majority of GABAA receptors in the mammalian brain consist of two α-, two ß- and one γ- or δ-subunit. This subunit organization crucially determines the physiological and pharmacological properties of the GABAA receptors. Using immunohistochemistry, we investigated the distribution of 10 GABAA receptor subunits (α1, α2, α3, α4, α5, ß1, ß2, ß3, γ2, and δ) in the fore brain of three female rhesus monkeys (Macaca mulatta). Within the cerebral cortex, subunits α1, α5, ß2, ß3, and γ2 were found in all layers, α2, α3, and ß1 were more concentrated in the inner and outer layers. The caudate/putamen was rich in α1, α2, α5, all three ß-subunits, γ2, and δ. Subunits α3 and α5 were more concentrated in the caudate than in the putamen. In contrast, α1, α2, ß1, ß2, γ2, and δ were highest in the pallidum. Most dorsal thalamic nuclei contained subunits α1, α2, α4, ß2, ß3, and γ2, whereas α1, α3, ß1, and γ2 were most abundant in the reticular nucleus. Within the amygdala, subunits α1, α2, α5, ß1, ß3, γ2, and δ were concentrated in the cortical nucleus, whereas in the lateral and basolateral amygdala α1, α2, α5, ß1, ß3, and δ, and in the central amygdala α1, α2, ß3, and γ2 were most abundant. Interestingly, subunit α3-IR outlined the intercalated nuclei of the amygdala. In the hippocampus, subunits α1, α2, α5, ß2, ß3, γ2, and δ were highly expressed in the dentate molecular layer, whereas α1, α2, α3, α5, ß1, ß2, ß3, and γ2 were concentrated in sector CA1 and the subiculum. The distribution of GABAA receptor subunits in the rhesus monkey was highly heterogeneous indicating a high number of differently assembled receptors. In most areas investigated, notably in the striatum/pallidum, amygdaloid nuclei and in the hippocampus it was more diverse than in the rat and mouse indicating a more heterogeneous and less defined receptor assembly in the monkey than in rodent brain.

Sonic hedgehog expression in zebrafish forebrain identifies the teleostean pallidal signaling center and shows preglomerular complex and posterior tubercular dopamine cells to arise from shh cells.

Ventralization, a major patterning process in the developing vertebrate neural tube (central nervous system, CNS), depends on Sonic hedgehog (SHH) as a main signaling morphogen. We studied the CNS of late larval and young adult zebrafish in a transgenic shh-GFP line revealing increased neuroanatomical detail due to the progressed differentiation state compared to earlier stages. Some major findings emerge from the present study. (a) shh -GFP is still expressed along the adult zebrafish CNS neuraxis in most locations seen in larvae. (b) We newly identify a ventroposterior shh pallidal domain representing the basal telencephalic signaling center important for basal ganglia development known in other vertebrates (i.e., the anterior entopeduncular area-basal medial ganglionic eminence of mammals). (c) We further show late-emerging shh-GFP positive radial glia cells in the medial zone of the dorsal telencephalon (i.e., the teleostan pallial amygdala). (d) Immunostains for tyrosine hydroxylase demonstrate that there is selective colocalization in adult dopamine cells with shh-GFP in the posterior tuberculum, including in projection cells to striatum, which represents a striking parallel to amniote mesodiencephalic dopamine cell origin from shh expressing floor plate cells. (e) There is no colocalization of shh and islet1 as shown by respective shh-GFP and islet1-GFP lines. (f) The only radially far migrated shh-GFP cells are located in the preglomerular area. (g) There are no adult cerebellar and tectal shh-GFP cells confirming their exclusive role during early development as previously reported by our laboratory.

Search for appropriate reference genes for quantitative reverse transcription PCR studies in somite, prosencephalon and heart of early mouse embryo.

qRT-PCR requires reliable internal control genes stably expressed in different samples and experimental conditions. The stability of reference genes is rarely tested experimentally, especially in developing tissues given the singularity of these samples. Here we evaluated the suitability of a set of reference genes (Actb, Gapdh, Tbp, Pgk1 and Sdha) using samples from early mouse embryo tissues that are widely used in research (somites, prosencephalon and heart) at different developmental stages. The comparative ΔCq method and five software packages (NormFinder, geNorm, BestKeeper, DataAssist and RefFinder) were used to rank the most stable genes while GenEx and GeNorm programs determined the optimal total number of reference genes for a reliable normalization. The ranking of most reliable reference genes was different for each tissue evaluated: (1) in somite from embryos with 16-18 somite pairs stage, the combination of Pgk1 and Actb provided the best normalization and Actb also presented high stability levels at an earlier developmental stage; (2) Gapdh is the most stable gene in prosencephalon in the two developmental stages tested; and (3) in heart samples, Sdha, Gapdh and Actb were the best combination for qPCR normalization. The analysis of these three tissues simultaneously indicated the combination of Gapdh, Actb and Tbp as the most reliable internal control. This study highlights the importance of appropriate reference genes according to the cell type and/or tissue of interest. The data here described can be applied in future research using mouse embryos as a model for mammalian development.

Sestd1 Encodes a Developmentally Dynamic Synapse Protein That Complexes With BCR Rac1-GAP to Regulate Forebrain Dendrite, Spine and Synapse Formation.

SEC14 and Spectrin domain-1 (Sestd1) is a synapse protein that exhibits a striking shift from the presynaptic to postsynaptic space as neurons mature postnatally in the mouse hippocampus. Hippocampal pyramidal neurons from mice with global genetic deletion of Sestd1 have reduced dendrite arbors, spines, and excitatory synapses. Electrophysiologically this correlates with cell-autonomous reductions in both AMPA- and NMDA-excitatory postsynaptic currents in individual hippocampal neurons from which Sestd1 has been deleted in vivo. These neurodevelopmental and functional deficits are associated with increased activation of the Rho family GTPases Rac1 and RhoA. Co-immunoprecipitation and mass spectrometry reveal that the Breakpoint Cluster Region protein, a Rho GTPase activating protein (GAP), forms complexes with Sestd1 in brain tissue. This complements earlier findings that Sestd1 can also partner with other Rho family GAPs and guanine nucleotide exchange factors. Our findings demonstrate that Sestd1 is a developmentally dynamic synaptic regulator of Rho GTPases that contributes to dendrite and excitatory synapse formation within differentiating pyramidal neurons of the forebrain.

[N-WASP regulates cortical neuron migration through its polyPro and VCA domains].

Cortical neuron migration in the developing mouse forebrain is a complex process, which contains several steps related to cytoskeleton dynamics and remodeling. Neural Wiskott-Aldrich syndrome protein (N-WASP), a member of the WASP-WAVE family, regulates actin cytoskeleton reorganization through the binding of its VCA domain to the Arp2/3 complex. Here we report expression patterns of N-WASP gene in the mouse developing embryonic cortex (E12.5~ E18.5) and find its expression levels are decreased during embryonic development. By using in utero electroporation (IUE) method, we find that either N-WASP overexpression or knockdown impairs cortical neuron migration, and the defects of cortical neuron migration caused by N-WASP overexpression are much more severe than that by its knockdown. N-WASP protein contains four domains: WH1, GBD, polyPro, and VCA. We generated a series of dominant negative N-WASP mutants by modifying these domains. Overexpression of N-WASP mutant lacking domain polyPro, VCA, or WH1, impairs cortical neuron migration. However, overexpression of N-WASP with the H208D point mutation, which abolishes the Cdc42 binding to N-WASP, causes only a marginal defect of cortical neuron migration. Finally, overexpression of the individual domain polyPro or VCA, but not WH1, can recapitulate the defects by N-WASP overexpression. However, overexpression of WH1-GBD fragment has no apparent effect on cortical neuron migration. In conclusion, our data demonstrate that N-WASP regulates cortical neuron migration mainly through its polyPro and VCA domains.

Quantitative mapping reveals age and sex differences in vasopressin, but not oxytocin, immunoreactivity in the rat social behavior neural network.

The neuropeptides vasopressin (AVP) and oxytocin (OT) have been implicated in the regulation of numerous social behaviors in adult and juvenile animals. AVP and OT signaling predominantly occur within a circuit of interconnected brain regions known collectively as the "social behavior neural network" (SBNN). Importantly, AVP and OT signaling within the SBNN has been shown to differentially regulate diverse social behaviors, depending on the age and/or sex of the animal. We hypothesized that variation in the display of these behaviors is due in part to age and sex differences in AVP and OT synthesis within the SBNN. However, a thorough characterization of AVP and OT-immunoreactive (ir) fibers and cell bodies across age and sex within the SBNN has been lacking in rats. We therefore quantified AVP- and OT-ir fibers and cell bodies in 22 subregions of the forebrain SBNN in juvenile and adult, male and female rats. We found numerous age (16 subregions) and sex (10 subregions) differences in AVP-ir fiber fractional areas, and AVP-ir cell body numbers, which were mainly observed in the medial amygdala/bed nucleus of the stria terminalis to lateral septum circuit. In contrast to AVP, we observed no age or sex differences in OT-ir fiber fractional areas or cell bodies in any of the 22 subregions of the forebrain SBNN. Thus, unlike the static pattern observed for OT, AVP innervation of the forebrain SBNN appears to undergo developmental changes, and is highly sexually dimorphic, which likely has significant functional consequences for the regulation of social behavior.

Effects of Acyzol on Zinc Content in Rat Brain and Blood Plasma.

Zinc level in the blood plasma and brain of rats was studied by inductively coupled plasma mass spectrometry. Maximum amount of zinc was observed in the cerebellum (15.0±5.5 µg/mg wet tissue). Single intraperitoneal administration of a zinc donor acyzol (24 mg/kg) did not change the content of this element in the tissues. Repeated injections of acyzol (7 injections over 14 days) significantly increased zinc level in rat plasma and brain. This elevation was most pronounced in the forebrain (cortex and subcortical structures). The rise in zinc concentration in blood plasma correlated with its level in the brain.

Targeted Antioxidant, Catalase-SKL, Reduces Beta-Amyloid Toxicity in the Rat Brain.

Accumulation of beta-amyloid (Aß) in the brain has been implicated as a major contributor to the cellular pathology and cognitive impairment observed in Alzheimer's disease. Beta-amyloid may exert its toxic effects by increasing reactive oxygen species and neuroinflammation in the brain. This study set out to investigate whether a genetically engineered derivative of the peroxisomal antioxidant enzyme catalase (CAT-SKL), is able to reduce the toxicity induced by intracerebroventricular injection of Aß25-35 in the mature rat brain. Histopathological and immunohistochemical analyses were used to evaluate neuroinflammation, and neuronal loss. Spatial learning and reference memory was assessed using the Morris water maze. CAT-SKL treatment was able to reduce the pathology induced by Aß25-35 toxicity by significantly decreasing microglia activation in the basal forebrain and thalamus, and reducing cholinergic loss in the basal forebrain. Aß25-35 animals showed deficits in long-term reference memory in the Morris water maze, while Aß25-35 animals treated with CAT-SKL did not demonstrate long-term memory impairments. This preclinical data provides support for the use of CAT-SKL in reducing neuroinflammation and long-term reference memory deficits induced by Aß25-35.

Increased complexity of circRNA expression during species evolution.

Circular RNAs (circRNAs) are broadly identified from precursor mRNA (pre-mRNA) back-splicing across various species. Recent studies have suggested a cell-/tissue- specific manner of circRNA expression. However, the distinct expression pattern of circRNAs among species and its underlying mechanism still remain to be explored. Here, we systematically compared circRNA expression from human and mouse, and found that only a small portion of human circRNAs could be determined in parallel mouse samples. The conserved circRNA expression between human and mouse is correlated with the existence of orientation-opposite complementary sequences in introns that flank back-spliced exons in both species, but not the circRNA sequences themselves. Quantification of RNA pairing capacity of orientation-opposite complementary sequences across circRNA-flanking introns by Complementary Sequence Index (CSI) identifies that among all types of complementary sequences, SINEs, especially Alu elements in human, contribute the most for circRNA formation and that their diverse distribution across species leads to the increased complexity of circRNA expression during species evolution. Together, our integrated and comparative reference catalog of circRNAs in different species reveals a species-specific pattern of circRNA expression and suggests a previously under-appreciated impact of fast-evolved SINEs on the regulation of (circRNA) gene expression.

Proteomic analysis of protein composition of rat forebrain cortex exposed to morphine for 10days; comparison with animals exposed to morphine and subsequently nurtured for 20days in the absence of this drug.

UNLABELLED: Proteomic analysis was performed in post-nuclear supernatant fraction (PNS) prepared from forebrain cortex of rats exposed to increasing doses of morphine (10-50mg/kg) for 10days and sacrificed 24h (group +M10) or 20days (group +M10/-M20) after the last dose of morphine. PNS fraction was resolved by 2D-ELFO and stained by CBB. Analysis of the difference between (+M10) and (-M10) samples of PNS by PDQuest accompanied by MALDI-TOF MS/MS indicated the significant change of 28 proteins. Importantly, the number of altered proteins was decreased to 14 after 20days of nurturing animals in the absence of morphine. This new and important finding indicating the ability of mammalian organism to return to physiological norm after removal of the drug was verified by an independent methodology - gel-free & label-free quantification and normalization procedure denominated as MaxLFQ. The 113 proteins were identified as altered by morphine in (+M10) samples when compared with (-M10) samples of PNS and this number was decreased to 19 after 20days of nurturing the animals in the absence of this drug. BIOLOGICAL SIGNIFICANCE: Forebrain cortex of rats exposed to morphine for 10days is severely altered as far as the overall protein composition is involved. Depending on the method used for protein detection and quantification, 28 (MALDI-TOF MS/MS) or 113 (MaxLFQ) altered proteins were identified. Importantly, in rats sacrificed 20days after the last dose of morphine, the number of altered proteins was decreased to 14 (MALDI-TOF MS/MS) and 19 (MaxLFQ), respectively. Our data indicate the high ability of living organism to oppose the drastic, morphine-induced change of the target tissue protein composition with the aim to return to the physiological norm after complete removal of the drug.

Forebrain-Specific Loss of BMPRII in Mice Reduces Anxiety and Increases Object Exploration.

To investigate the role of Bone Morphogenic Protein Receptor Type II (BMPRII) in learning, memory, and exploratory behavior in mice, a tissue-specific knockout of BMPRII in the post-natal hippocampus and forebrain was generated. We found that BMPRII mutant mice had normal spatial learning and memory in the Morris water maze, but showed significantly reduced swimming speeds with increased floating behavior. Further analysis using the Porsolt Swim Test to investigate behavioral despair did not reveal any differences in immobility between mutants and controls. In the Elevated Plus Maze, BMPRII mutants and Smad4 mutants showed reduced anxiety, while in exploratory tests, BMPRII mutants showed more interest in object exploration. These results suggest that loss of BMPRII in the mouse hippocampus and forebrain does not disrupt spatial learning and memory encoding, but instead impacts exploratory and anxiety-related behaviors.

Distinct brainstem and forebrain circuits receiving tracheal sensory neuron inputs revealed using a novel conditional anterograde transsynaptic viral tracing system.

Sensory nerves innervating the mucosa of the airways monitor the local environment for the presence of irritant stimuli and, when activated, provide input to the nucleus of the solitary tract (Sol) and paratrigeminal nucleus (Pa5) in the medulla to drive a variety of protective behaviors. Accompanying these behaviors are perceivable sensations that, particularly for stimuli in the proximal end of the airways, can be discrete and localizable. Airway sensations likely reflect the ascending airway sensory circuitry relayed via the Sol and Pa5, which terminates broadly throughout the CNS. However, the relative contribution of the Sol and Pa5 to these ascending pathways is not known. In the present study, we developed and characterized a novel conditional anterograde transneuronal viral tracing system based on the H129 strain of herpes simplex virus 1 and used this system in rats along with conventional neuroanatomical tracing with cholera toxin B to identify subcircuits in the brainstem and forebrain that are in receipt of relayed airway sensory inputs via the Sol and Pa5. We show that both the Pa5 and proximal airways disproportionately receive afferent terminals arising from the jugular (rather than nodose) vagal ganglia and the output of the Pa5 is predominately directed toward the ventrobasal thalamus. We propose the existence of a somatosensory-like pathway from the proximal airways involving jugular ganglia afferents, the Pa5, and the somatosensory thalamus and suggest that this pathway forms the anatomical framework for sensations arising from the proximal airway mucosa.

Detergent-dependent separation of postsynaptic density, membrane rafts and other subsynaptic structures from the synaptic plasma membrane of rat forebrain.

We systematically investigated the purification process of post-synaptic density (PSD) and post-synaptic membrane rafts (PSRs) from the rat forebrain synaptic plasma membranes by examining the components and the structures of the materials obtained after the treatment of synaptic plasma membranes with TX-100, n-octyl ß-d-glucoside (OG) or 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO). These three detergents exhibited distinct separation profiles for the synaptic subdomains. Type I and type II PSD proteins displayed mutually exclusive distribution. After TX-100 treatment, type I PSD was recovered in two fractions: a pellet and an insoluble fraction 8, which contained partially broken PSD-PSR complexes. Conventional PSD was suggested to be a mixture of these two PSD pools and did not contain type II PSD. An association of type I PSD with PSRs was identified in the TX-100 treatment, and those with type II PSD in the OG and CHAPSO treatments. An association of GABA receptors with gephyrin was easily dissociated. OG at a high concentration solubilized the type I PSD proteins. CHAPSO treatment resulted in a variety of distinct fractions, which contained certain novel structures. Two different pools of GluA, either PSD or possibly raft-associated, were identified in the OG and CHAPSO treatments. These results are useful in advancing our understanding of the structural organization of synapses at the molecular level. We systematically investigated the purification process of post-synaptic density (PSD) and synaptic membrane rafts by examining the structures obtained after treatment of the SPMs with TX-100, n-octyl ß-d-glucoside or CHAPSO. Differential distribution of type I and type II PSD, synaptic membrane rafts, and other novel subdomains in the SPM give clues to understand the structural organization of synapses at the molecular level.

D-serine and serine racemase are localized to neurons in the adult mouse and human forebrain.

D-Serine, a co-agonist at the NMDA receptor (NMDAR), is synthesized from L-serine by the enzyme serine racemase (SR), which is heavily expressed in the forebrain. Although SR was originally reported to be localized exclusively to astrocytes, recent conditional knock out results demonstrate that little SR is expressed in forebrain astrocytes. As a consequence, the cellular location of its product, D-serine, in the brain is also uncertain. Immunocytochemistry now indicates that SR is expressed primarily in forebrain glutamatergic neurons with the remainder in GABAergic interneurons. We utilized SR deficient (SR-/-) mice, which have <15 % of normal D-serine levels, to validate and optimize a D-serine immunohistochemical method. Nearly all of the D-serine in neocortex and hippocampus (HP) is found in neurons, with virtually no D-serine co-localizing with two astrocyte markers. Interestingly, only a subset of the D-serine positive neurons contained SR in the neocortex and HP. Greater than half of the D-serine positive neurons were GABAergic interneurons, with a majority of these neurons containing parvalbumin and/or somatostatin. Only ~25-40 % of interneurons expressed SR in the neocortex and HP. Finally, we demonstrate in human post-mortem neocortex that SR is found in both excitatory and inhibitory neurons, but not in S100ß-containing astrocytes. In sum, these findings conclusively demonstrate that the majority of D-serine is both synthesized and stored in neurons. It will be important to determine the functional significance for the separation of synthesis and storage of D-serine in neurons, as well as the presence of this NMDAR co-agonist in GABAergic interneurons.

Distribution of secretin receptors in the rat central nervous system: an in situ hybridization study.

Secretin shows a wide distribution in the brain. Functional significance of central secretin is stressed since it has been associated with autism and schizophrenia. The presence of the secretin receptor was previously demonstrated in the brain by different methods. Neurons in the cerebellum, hypothalamic paraventricular and supraoptic nuclei, and in the vascular organ of lamina terminalis were shown to express secretin receptor mRNA by using in situ hybridization with digoxigenin-labeled probe. In this work, we used a very sensitive radioactive in situ hybridization technique and systematically mapped the expression of secretin receptor mRNA in the brain. The densest labeling was observed in the nucleus of solitary tract and in the laterodorsal thalamic nucleus, where decreasing number of receptors was seen in the vascular organ of lamina terminalis, and the lateral habenular complex, and then in the supraoptic nucleus. Only a few scattered labeled cells were observed in the median frontal gyrus, entorhinal cortex, hypothalamic paraventricular nucleus, perifornical region, lateral hypothalamic area, head of the caudate nucleus, spinal trigeminal nucleus, and cerebellum. Secretin receptor mRNA showed a far wider distribution than was known before, suggesting a more significant functional relevance than thought earlier.

Preparing synaptoneurosomes from adult mouse forebrain.

Many neuroscience studies involve subcellular fractionation to produce isolated or enriched synaptic fractions. Synaptosomes are prepared by flotation of synaptic membranes on sucrose or Percoll gradients. Alternatively, synaptoneurosomes are prepared by filtration of tissue homogenate through a series of filters to obtain a fraction that is enriched in pinched-off dendritic spines. Whereas the protocol for making synaptosomes is reasonably well standardized and well described in the literature, there is (to our knowledge) no detailed lab protocol for making synaptoneurosomes. Here, we give the methods used in our laboratory to produce synaptoneurosomes that are suitable for studying RNAs and proteins.