Phenotypic differences in hiPSC NPCs derived from patients with schizophrenia.

Consistent with recent reports indicating that neurons differentiated in vitro from human-induced pluripotent stem cells (hiPSCs) are immature relative to those in the human brain, gene expression comparisons of our hiPSC-derived neurons to the Allen BrainSpan Atlas indicate that they most resemble fetal brain tissue. This finding suggests that, rather than modeling the late features of schizophrenia (SZ), hiPSC-based models may be better suited for the study of disease predisposition. We now report that a significant fraction of the gene signature of SZ hiPSC-derived neurons is conserved in SZ hiPSC neural progenitor cells (NPCs). We used two independent discovery-based approaches-microarray gene expression and stable isotope labeling by amino acids in cell culture (SILAC) quantitative proteomic mass spectrometry analyses-to identify cellular phenotypes in SZ hiPSC NPCs from four SZ patients. From our findings that SZ hiPSC NPCs show abnormal gene expression and protein levels related to cytoskeletal remodeling and oxidative stress, we predicted, and subsequently observed, aberrant migration and increased oxidative stress in SZ hiPSC NPCs. These reproducible NPC phenotypes were identified through scalable assays that can be applied to expanded cohorts of SZ patients, making them a potentially valuable tool with which to study the developmental mechanisms contributing to SZ.

Morphometric characterization of synapses in the primate prefrontal cortex formed by afferents from the mediodorsal thalamic nucleus.

The main thalamic afferentation of the prefrontal cortex (PFC) originates in the mediodorsal nucleus (MD). Although it is suggested that this pathway is affected in schizophrenia, there is a lack of functional and structural data regarding its synaptic organization. The scope of this study was to characterize the ultrastructural features of thalamocortical synapses formed by afferents from the MD by applying anterograde tract tracing, immunohistochemical detection of parvalbumin (PV, a probable marker of thalamocortical endings), and quantitative electron microscopic techniques to the PFC of the macaque monkey. Our findings indicate that anterogradely-labeled and PV-immunoreactive boutons exhibit similar ultrastructural properties, characterized by their larger size, higher incidence of release sites and a higher occurrence of mitochondria when compared to non-labeled, excitatory-like endings in the middle layers of the PFC. Although most of the contacts were made on spines in both cases, PV-immunopositive axon terminals apparently targeted dendritic shafts at about twice the frequency found for anterogradely-labeled afferents from the MD (20.5% and 9.5%, respectively). This result suggests diversity among thalamocortical and/or PV-immunoreactive axon terminals of the PFC. In accordance with studies in other cortical areas, our findings suggest that corollary discharge through the mediodorsal thalamocortical projection is also adapted to synaptic transmission with high efficacy and probably exhibits marked short-term temporal dynamics in the PFC.

Differential anterior prefrontal activation during the recognition stage of a spatial working memory task.

Neuroimaging studies commonly show widespread activations in the prefrontal cortex during various forms of working memory and long-term memory tasks. However, the anterior prefrontal cortex (aPFC, Brodmann area 10) has been mainly associated with retrieval in episodic memory, and its role in working memory is less clear. We conducted an event-related functional magnetic resonance imaging study to examine brain activations in relation to recognition in a spatial delayed-recognition task. Similar to the results from previous findings, several frontal areas were strongly activated during the recognition phase of the task, including the aPFC, the lateral PFC and the anterior cingulate cortex. Although the aPFC was more active during the recognition phase, it was also active during the delay phase of the spatial working memory task. In addition, the aPFC showed greater activity in response to negative probes (non-targets) than to positive probes (targets). While our analyses focused on examining signal changes in the aPFC, other prefrontal regions showed similar effects and none of the areas were more active in response to the positive probes than to the negative probes. Our findings support the conclusion that the aPFC is involved in working memory and particularly in processes that distinguish target and non-target stimuli during recognition.

Division of labor among distinct subtypes of inhibitory neurons in a cortical microcircuit of working memory.

A conspicuous feature of cortical organization is the wide diversity of inhibitory interneurons; their differential computational functions remain unclear. Here we propose a local cortical circuit in which three major subtypes of interneurons play distinct roles. In a model designed for spatial working memory, stimulus tuning of persistent activity arises from the concerted action of widespread inhibition mediated by perisoma-targeting (parvalbumin-containing) interneurons and localized disinhibition of pyramidal cells via interneuron-targeting (calretinin-containing) interneurons. Moreover, resistance against distracting stimuli (a fundamental property of working memory) is dynamically controlled by dendrite-targeting (calbindin-containing) interneurons. The experimental observation of inverted tuning curves of monkey prefrontal neurons recorded during working memory supports a key model prediction. This work suggests a framework for understanding the division of labor and cooperation among different inhibitory cell types in a recurrent cortical circuit.

Sustained mnemonic response in the human middle frontal gyrus during on-line storage of spatial memoranda.

The mapping of cognitive functions to neural systems is a central goal of cognitive neuroscience. On the basis of homology with lesion and physiological studies in nonhuman primates, Brodmann's area (BA) 46/9 in the middle frontal gyrus (MFG) has been proposed as the cortical focus for both the storage as well as processing components of working memory in the human brain, but the evidence on the segregation of these components and their exact areal localization has been inconsistent. In order to study this issue and increase the temporal resolution of functional mapping, we disambiguated the storage component of working memory from sensory and motor responses by employing functional magnetic resonance imaging (fMRI) in spatial delayed-response (DR) tasks with long delay intervals and different conditions of demand. We here show that BA 46 can support a sustained mnemonic response for as long as 24 sec in a high-demand task and the signal change in this area exceeded that in the other prefrontal areas examined. Our findings support a conservation of functional architecture between human and nonhuman primate in showing that the MFG is prominently engaged in the storage of spatial information.

Distribution of protein phosphatases-1 alpha and -1 gamma 1 and the D(1) dopamine receptor in primate prefrontal cortex: Evidence for discrete populations of spines.

The function of G protein-coupled receptors depends on the availability of the appropriate signal transduction proteins in close proximity to the receptor. We have examined and quantified in primate prefrontal cortex the subcellular distribution of two isoforms of protein phosphatase-1 (PP1), PP1 alpha and PP1 gamma 1, which are components of the signal transduction pathway accessed by the D(1) dopamine receptor. Both PP1 alpha- and PP1 gamma 1-labeled puncta are seen in cortex, basal ganglia, hippocampus, and thalamus. Viewed with the electron microscope, both PP1 isoforms are selectively localized to dendritic spines and are found in different percentages of spines; PP1 alpha is present in roughly 70% and PP1 gamma 1 in roughly 40% of dendritic spines. Our analysis indicates that three populations of spines are defined by the distribution of these PP1 isoforms: those that contain both PP1 alpha and PP1 gamma 1, those that contain only PP1 alpha and those that contain neither. The D(1) receptor is present in a subset of the population that contains both PP1 alpha and PP1 gamma 1. The nonhomogeneous distribution of signal transduction proteins in the spines and dendrites of cortical pyramidal cells may help to explain differences in the actions of receptors that nominally use the same signal-transduction pathway.

Cell proliferation without neurogenesis in adult primate neocortex.

A recent assertion that new neurons are continually added to the neocortex of adult macaque monkeys has profound implications for understanding the cellular mechanisms of higher cognitive functions. Here we searched for neurogenesis in adult macaques by using immunofluorescent triple labeling for the DNA-replication indicator, bromodeoxyuridine (BrdU), and neuronal and glial cell markers. Although numerous BrdU-labeled cells were distributed throughout the cerebral wall, including the neocortex, these were identified as nonneuronal cells; evidence for newly generated neurons was limited to the hippocampus and olfactory bulb. Thus, our results do not substantiate the claim of neurogenesis in normal adult primate neocortex.

Requirement of the JIP1 scaffold protein for stress-induced JNK activation.

The c-Jun N-terminal kinase (JNK) signal transduction pathway is activated in response to the exposure of cells to environmental stress. Components of the JNK signaling pathway interact with the JIP1 scaffold protein. JIP1 is located in the neurites of primary hippocampal neurons. However, in response to stress, JIP1 accumulates in the soma together with activated JNK and phosphorylated c-Jun. Disruption of the Jip1 gene in mice by homologous recombination prevented JNK activation caused by exposure to excitotoxic stress and anoxic stress in vivo and in vitro. These data show that the JIP1 scaffold protein is a critical component of a MAP-kinase signal transduction pathway.

Telencephalic origin of human thalamic GABAergic neurons.

In non-primate mammalian species, telencephalic and diencephalic neurons originate from their respective local proliferative zones. Using vital dye labeling in organotypic slice cultures, we show that in human brain, a contingent of neurons from the ganglionic eminence of the telencephalon migrate to the dorsal thalamic association nuclei of the diencephalon. These neurons rely on homotypic-neurophilic guidance during their migration, are GABAergic, and express Dlx1/2 homeodomain-containing proteins. Similar experiments in a non-human primate and in rodent embryos did not reveal a similar migratory pathway. Migration assays demonstrated that the human dorsal thalamus attracts telencephalic cells, an effect not observed in the mouse, in which such migration is inhibited by chemorepulsive cues. These data suggest that modifications in the pattern of migratory guidance cues in the forebrain may underlie the appearance of a new migratory pathway and thus contribute to the evolutionary expansion of the thalamic association nuclei in the human.

JNK3 contributes to c-Jun activation and apoptosis but not oxidative stress in nerve growth factor-deprived sympathetic neurons.

The stress activated protein kinase pathway culminates in c-Jun phosphorylation mediated by the Jun Kinases (JNKs). The role of the JNK pathway in sympathetic neuronal death is unclear in that apoptosis is not inhibited by a dominant negative protein of one JNK kinase, SEK1, but is inhibited by CEP-1347, a compound known to inhibit this overall pathway but not JNKs per se. To evaluate directly the apoptotic role of the JNK isoform that is selectively expressed in neurons, JNK3, we isolated sympathetic neurons from JNK3-deficient mice and quantified nerve growth factor (NGF) deprivation-induced neuronal death, oxidative stress, c-Jun phosphorylation, and c-jun induction. Here, we report that oxidative stress in neurons from JNK3-deficient mice is normal after NGF deprivation. In contrast, NGF-deprivation-induced increases in the levels of phosphorylated c-Jun, c-jun, and apoptosis are each inhibited in JNK3-deficient mice. Overall, these results indicate that JNK3 plays a critical role in activation of c-Jun and apoptosis in a classic model of cell-autonomous programmed neuron death.

Development of layer I neurons in the primate cerebral cortex.

Layer I, which plays an important role in the development of the cerebral cortex, expands in size and diversity in primates. We found that, unlike in rodents, in the macaque monkey, neurons of this layer are generated during the entire 2 month period of corticogenesis, within the middle of the 165-d-long gestation. The large, classical Cajal-Retzius cells, immunoreactive to reelin and calretinin but not to GABA, are generated first [embryonic day 38 (E38)-E50], with the peak of [(3)H]thymidine ([(3)H]TdR) labeling at E43. Ultrastructural analysis revealed that processes of these cells form a stereotyped, rectangular network oriented parallel to the pial surface. Genesis of smaller, GABAergic neurons begins slightly later (E43), reaches a peak of [(3)H]TdR labeling between E54 and E70, and continues until the completion of corticogenesis (E94). These late-generated layer I cells are imported from outside sources such as the olfactory primordium and ganglionic eminence and via a massive subpial granular layer that may also supply some GABAergic interneurons to the subjacent cortical plate. The ratio of large-to-small layer I neurons changes differentially, indicating that each class is produced and/or eliminated at a different rate and suggesting that their roles in primates are diverse.

Programmed cell death of developing mammalian neurons after genetic deletion of caspases.

An analysis of programmed cell death of several populations of developing postmitotic neurons after genetic deletion of two key members of the caspase family of pro-apoptotic proteases, caspase-3 and caspase-9, indicates that normal neuronal loss occurs. Although the amount of cell death is not altered, the death process may be delayed, and the cells appear to use a nonapoptotic pathway of degeneration. The neuronal populations examined include spinal interneurons and motor, sensory, and autonomic neurons. When examined at both the light and electron microscopic levels, the caspase-deficient neurons exhibit a nonapoptotic morphology in which nuclear changes such as chromatin condensation are absent or reduced; in addition, this morphology is characterized by extensive cytoplasmic vacuolization that is rarely observed in degenerating control neurons. There is also reduced terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling in dying caspase-deficient neurons. Despite the altered morphology and apparent temporal delay in cell death, the number of neurons that are ultimately lost is indistinguishable from that seen in control animals. In contrast to the striking perturbations in the morphology of the forebrain of caspase-deficient embryos, the spinal cord and brainstem appear normal. These results are consistent with the growing idea that the involvement of specific caspases and the occurrence of caspase-independent programmed cell death may be dependent on brain region, cell type, age, and species or may be the result of specific perturbations or pathology.

Primate rhinal cortex participates in both visual recognition and working memory tasks: functional mapping with 2-DG.

The rhinal cortex in the medial temporal lobe has been implicated in object recognition memory tasks and indeed is considered to be the critical node in a visual memory network. Previous studies using the 2-deoxyglucose method have shown that thalamic and hippocampal structures thought to be involved in visual recognition memory are also engaged by spatial and object working memory tasks in the nonhuman primate. Networks engaged in memory processing can be recognized by analysis of patterns of activation accompanying performance of specifically designed tasks. In the present study, we compared metabolic activation of the entorhinal and perirhinal cortex during the performance of three working memory tasks [delayed response (DR), delayed alternation (DA), and delayed object alternation (DOA)] to that induced by a standard recognition memory task [delayed match-to-sample (DMS)] and a sensorimotor control task in rhesus monkeys. A region-of-interest analysis revealed elevated local cerebral glucose utilization in the perirhinal cortex in animals performing the DA, DOA, and DMS tasks, and animals performing the DMS task were distinct in showing a strong focus of activation in the lateral perirhinal cortex. No significant differences were evident between groups performing memory and control tasks in the entorhinal cortex. These findings suggest that the perirhinal cortex may play a much broader role in memory processing than has been previously thought, encompassing explicit working memory as well as recognition memory.

Muscarinic m1 and m2 receptor proteins in local circuit and projection neurons of the primate striatum: anatomical evidence for cholinergic modulation of glutamatergic prefronto-striatal pathways.

The cellular and subcellular localization of muscarinic receptor proteins m1 and m2 was examined in the neostriatum of macaque monkeys by using light and electron microscopic immunocytochemical techniques. Double-labeling immunocytochemistry revealed m1 receptors in calbindin-D28k--positive medium spiny projection neurons. Muscarinic m1 labeling was dramatically more intense in the striatal matrix compartment in juvenile monkeys but more intense in striosomes in the adult caudate, suggesting that m1 expression undergoes a developmental age-dependent change. Ultrastructurally, m1 receptors were predominantly localized in asymmetric synapse-forming spines, indicating that these spines receive extrastriatal excitatory afferents. The association of m1-positive spines with lesion-induced degenerating prefronto-striatal axon terminals demonstrated that these afferents originate in part from the prefrontal cortex. The synaptic localization of m1 in these spines indicates a role of m1 in the modulation of excitatory neurotransmission. To a lesser extent, m1 was present in symmetric synapses, where it may also modulate inhibitory neurotransmission originating from local striatal neurons or the substantia nigra. Conversely, m2/choline acetyltransferase (ChAT) double labeling revealed that m2-positive neurons corresponded to large aspiny cholinergic interneurons and ultrastructurally, that the majority of m2 labeled axons formed symmetric synapses. The remarkable segregation of the m1 and m2 receptor proteins to projection and local circuit neurons suggests a functional segregation of m1 and m2 mediated cholinergic actions in the striatum: m1 receptors modulate extrinsic glutamatergic and monoaminergic afferents and intrinsic GABAergic afferents onto projection neurons, whereas m2 receptors regulate acetylcholine release from axons of cholinergic interneurons.

Prefrontal microcircuits: membrane properties and excitatory input of local, medium, and wide arbor interneurons.

To elucidate cortical mechanisms involved in higher cortical functions such as working memory, we have examined feedforward excitation transmitted by identified pyramidal cells to interneurons with predominantly horizontal axonal arbors, using dual somatic recordings in prefrontal cortical slices. Interneurons with local (narrow) axonal arbors, especially chandelier interneurons, exhibited extremely narrow action potentials and high evoked firing rates, whereas neurons identified with wide arbor axons generated wider spikes and lower evoked firing rates with considerable spike adaptation, resembling that of pyramidal cells. Full reconstruction of differentially labeled neuronal pairs revealed that local arbor cells generally received a single but functionally reliable putative synaptic input from the identified pyramidal neuron member of the pair. In contrast, more synapses (two to five) were necessary to depolarize medium and wide arbor neurons reliably. The number of putative synapses and the amplitude of the postsynaptic response were remarkably highly correlated within each class of local, medium, and wide arbor interneurons (r = 0.88, 0.95, and 0.99, respectively). Similarly strong correlations within these subgroups were also present between the number of putative synapses and variance in the EPSP amplitudes, supporting the validity of our morphological analysis. We conclude that interneurons varying in the span of their axonal arbors and hence in the potential regulation of different numbers of cortical modules differ also in their excitatory synaptic input and physiological properties. These findings provide insight into the circuit basis of lateral inhibition and functional interactions within and between cortical columns in the cerebral cortex.

Coding specificity in cortical microcircuits: a multiple-electrode analysis of primate prefrontal cortex.

Neurons with directional specificities are active in the prefrontal cortex (PFC) during tasks that require spatial working memory. Although the coordination of neuronal activity in PFC is thought to be maintained by a network of recurrent connections, direct physiological evidence regarding such networks is sparse. To gain insight into the functional organization of the working memory system in vivo, we recorded simultaneously from multiple neurons spaced 0.2-1 mm apart in monkeys performing an oculomotor delayed response task. We used cross-correlation analysis and characterized the effective connectivity between neurons in relation to their spatial and temporal response properties. The majority of narrow (<5 msec) cross-correlation peaks indicated common input and were most often observed between pairs of neurons within 0.3 mm of each other. Neurons recorded at these distances represented the full range of spatial locations, suggesting that the entire visual hemifield is represented in modules of corresponding dimensions. Nearby neurons could be activated in any epoch of the behavioral task (stimulus presentation, delay, response). The incidence and strength of cross-correlation, however, was highest among cells sharing similar spatial tuning and similar temporal profiles of activation across task epochs. The dependence of correlated discharge on the functional properties of neurons was observed both when we analyzed firing from the task period as well as from baseline fixation. Our results suggest that the coding specificity of individual neurons extends to the local circuits of which they are part.

Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A.

We have used a yeast two-hybrid approach to uncover protein interactions involving the D2-like subfamily of dopamine receptors. Using the third intracellular loop of the D2S and D3 dopamine receptors as bait to screen a human brain cDNA library, we identified filamin A (FLN-A) as a protein that interacts with both the D2 and D3 subtypes. The interaction with FLN-A was specific for the D2 and D3 receptors and was independently confirmed in pull-down and coimmunoprecipitation experiments. Deletion mapping localized the dopamine receptor-FLN-A interaction to the N-terminal segment of the D2 and D3 dopamine receptors and to repeat 19 of FLN-A. In cultures of dissociated rat striatum, FLN-A and D2 receptors colocalized throughout neuronal somata and processes as well as in astrocytes. Expression of D2 dopamine receptors in FLN-A-deficient M2 melanoma cells resulted in predominant intracellular localization of the D2 receptors, whereas in FLN-A-reconstituted cells, the D2 receptor was predominantly localized at the plasma membrane. These results suggest that FLN-A may be required for proper cell surface expression of the D2 dopamine receptors. Association of D2 and D3 dopamine receptors with FLN-A provides a mechanism whereby specific dopamine receptor subtypes may be functionally linked to downstream signaling components via the actin cytoskeleton.

The generation, migration, and differentiation of olfactory neurons in the adult primate brain.

In adult rodents, neural progenitor cells in the subependymal (SZ) zone of the lateral cerebral ventricle generate neuroblasts that migrate in chains via the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into interneurons. However, the existence of this neurogenic migratory system in other mammals has remained unknown. Here, we report the presence of a homologue of the rodent SZ/RMS in the adult macaque monkey, a nonhuman Old World primate with a relatively smaller OB. Our results-obtained by using combined immunohistochemical detection of a marker for DNA replication (5-bromodeoxyuridine) and several cell type-specific markers-indicate that dividing cells in the adult monkey SZ generate neuroblasts that undergo restricted chain migration over an extended distance of more than 2 cm to the OB and differentiate into granule interneurons. These findings in a nonhuman primate extend and support the use of the SZ/RMS as a model system for studying neural regenerative mechanisms in the human brain.