Neurons with choline acetyltransferase immunoreactivity and mRNA are present in the human cerebral cortex

 We examined the cerebral cortex of five autopsied individuals without neurological and psychiatric diseases by immunohistochemistry using an anti-human recombinant choline acetyltransferase (ChAT) polyclonal antibody and in situ hybridization with ³⁵S-labeled human ChAT riboprobes. The immunohistochemistry detected positive neurons which were medium-sized or large pyramidal neurons located predominantly in layers III and V. The density of such neurons was higher in the motor and secondary sensory areas than in other cortical areas; the immunoreactive neurons in layer V were more densely distributed in the motor area and those in layer III were distributed in the secondary sensory areas. Positively stained, non-pyramidal neurons were observed in the superficial layer of the cingulate gyrus and parahippocampus. No immunoreactive neurons were found in the primary sensory areas. The in situ hybridization detected some neurons with signals for ChAT mRNA in the cerebral cortex, most of which were distributed in layer V of the motor area and in layer III of the secondary visual area. These results indicate that the human cerebral cortex contains cholinergic neurons and displays regional and laminal variations in their distribution. Accepted: 17 November 1998     

Close homolog of L1 modulates area-specific neuronal positioning and dendrite orientation in the cerebral cortex

We show that the neural cell recognition molecule Close Homolog of L1 (CHL1) is required for neuronal positioning and dendritic growth of pyramidal neurons in the posterior region of the developing mouse neocortex. CHL1 was expressed in pyramidal neurons in a high-caudal to low-rostral gradient within the developing cortex. Deep layer pyramidal neurons of CHL1-minus mice were shifted to lower laminar positions in the visual and somatosensory cortex and developed misoriented, often inverted apical dendrites. Impaired migration of CHL1-minus cortical neurons was suggested by strikingly slower rates of radial migration in cortical slices, failure to potentiate integrin-dependent haptotactic cell migration in vitro, and accumulation of migratory cells in the intermediate and ventricular/subventricular zones in vivo. The restriction of CHL1 expression and effects of its deletion in posterior neocortical areas suggests that CHL1 may regulate area-specific neuronal connectivity and, by extension, function in the visual and somatosensory cortex.     

Adenovirus-mediated WGA gene delivery for transsynaptic labeling of mouse olfactory pathways

Detailed knowledge of neuronal connectivity patterns is indispensable for studies of various aspects of brain functions. We previously established a genetic strategy for visualization of multisynaptic neural pathways by expressing wheat germ agglutinin (WGA) transgene under the control of neuron type-specific promoter elements in transgenic mice and Drosophila. In this paper, we have developed a WGA-expressing recombinant adenoviral vector system and applied it for analysis of the olfactory system. When the WGA-expressing adenovirus was infused into a mouse nostril, various types of cells throughout the olfactory epithelium were infected and expressed WGA protein robustly. WGA transgene products in the olfactory sensory neurons were anterogradely transported along their axons to the olfactory bulb and transsynaptically transferred in glomeruli to dendrites of the second-order neurons, mitral and tufted cells. WGA protein was further conveyed via the lateral olfactory tract to the olfactory cortical areas including the anterior olfactory nucleus, olfactory tubercle, piriform cortex and lateral entorhinal cortex. In addition, transsynaptic retrograde labeling was observed in cholinergic neurons in the horizontal limb of diagonal band, serotonergic neurons in the median raphe nucleus, and noradrenergic neurons in the locus coeruleus, all of which project centrifugal fibers to the olfactory bulb. Thus, the WGA-expressing adenovirus is a useful and powerful tool for tracing neural pathways and could be used in animals that are not amenable to the transgenic technology.     

Etiology of a genetically complex seizure disorder in Celf4 mutant mice

Mice deficient for the gene encoding the RNA-binding protein CELF4 (CUGBP, ELAV-like family member 4) have a complex seizure phenotype that includes both convulsive and non-convulsive seizures, depending upon gene dosage and strain background, modeling genetically complex epilepsy. Invertebrate CELF is associated with translational control in fruit fly ovary epithelium and with neurogenesis and neuronal function in the nematode. Mammalian CELF4 is expressed widely during early development, but is restricted to the central nervous system in adults. To better understand the etiology of the seizure disorder of Celf4 deficient mice, we studied seizure incidence with spatial and temporal conditional knockout Celf4 alleles. For convulsive seizure phenotypes, it is sufficient to delete Celf4 in adulthood at the age of 7 weeks. This timing is in contrast to absence-like non-convulsive seizures, which require deletion before the end of the first postnatal week. Interestingly, selective deletion of Celf4 from cerebral cortex and hippocampus excitatory neurons, but not from inhibitory neurons, is sufficient to lower seizure threshold and to promote spontaneous convulsions. Correspondingly, Celf4 deficient mice have altered excitatory, but not inhibitory, neurotransmission as measured by patch-clamp recordings of cortical layer V pyramidal neurons. Finally, immunostaining in conjunction with an inhibitory neuron-specific reporter shows that CELF4 is expressed predominantly in excitatory neurons. Our results suggest that CELF4 plays a specific role in regulating excitatory neurotransmission. We posit that altered excitatory neurotransmission resulting from Celf4 deficiency underlies the complex seizure disorder in Celf4 mutant mice.     

Guidance of cortical radial migration by gradient of diffusible factors

During the development of cerebral cortex, newborn pyramidal neurons originated from the ventricle wall migrate outwardly to the superficial layer of cortex under the guidance of radial glial filaments. Whether this radial migration of young neurons is guided by gradient of diffusible factors or simply driven by a mass action of newly generated neurons at the ventricular zone is entirely unknown, a potential guidance mechanism that has long been overlooked. Our recent study showed that a guidance molecule semaphorin-3A, which is expressed in descending gradient across cortical layers, may serve as a chemoattractive guidance signal for radial migration of newborn cortical neurons toward upper layers. We hypothesize the existence of four groups of extracellular factors that can guide the radial migration of young neurons: (1) attractive factors expressing in superficial layers of cortex, (2) repulsive factors enriched in the ventricular zone, (3) pro-migratory factors uniformly expressed in all cortical layers, and (4) stop signals locally expressed in the outmost layer of cortex.     

Gain modulation by nicotine in macaque v1

Acetylcholine is a ubiquitous cortical neuromodulator implicated in cognition. In order to understand the potential for acetylcholine to play a role in visual attention, we studied nicotinic acetylcholine receptor (nAChR) localization and function in area V1 of the macaque. We found nAChRs presynaptically at thalamic synapses onto excitatory, but not inhibitory, neurons in the primary thalamorecipient layer 4c. Furthermore, consistent with the release enhancement suggested by this localization, we discovered that nicotine increases responsiveness and lowers contrast threshold in layer 4c neurons. We also found that nAChRs are expressed by GABAergic interneurons in V1 but rarely by pyramidal neurons, and that nicotine suppresses visual responses outside layer 4c. All sensory systems incorporate gain control mechanisms, or processes which dynamically alter input/output relationships. We demonstrate that at the site of thalamic input to visual cortex, the effect of this nAChR-mediated gain is an enhancement of the detection of visual stimuli.     

Reliability and representational bandwidth in the auditory cortex

It is unclear why there are so many more neurons in sensory cortex than in the sensory periphery. One possibility is that these "extra" neurons are used to overcome cortical noise and faithfully represent the acoustic stimulus. Another possibility is that even after overcoming cortical noise, there is "excess representational bandwidth" available and that this bandwidth is used to represent conjunctions of auditory and nonauditory information for computation. Here, we discuss recent data about neuronal reliability in auditory cortex showing that cortical noise may not be as high as was previously believed. Although at present, the data suggest that auditory cortex neurons can be more reliable than those in the visual cortex, we speculate that the principles governing cortical computation are universal and that visual and other cortical areas can also exploit strategies based on similarly high-fidelity activity.     

Sensory experience restructures thalamocortical axons during adulthood

The brain's capacity to rewire is thought to diminish with age. It is widely believed that development stabilizes the synapses from thalamus to cortex and that adult experience alters only synaptic connections between cortical neurons. Here we show that thalamocortical (TC) inputs themselves undergo massive plasticity in adults. We combined whole-cell recording from individual thalamocortical neurons in adult rats with a recently developed automatic tracing technique to reconstruct individual axonal trees. Whisker trimming substantially reduced thalamocortical axon length in barrel cortex but not the density of TC synapses along a fiber. Thus, sensory experience alters the total number of TC synapses. After trimming, sensory stimulation evoked more tightly time-locked responses among thalamorecipient layer 4 cortical neurons. These findings indicate that thalamocortical input itself remains plastic in adulthood, raising the possibility that the axons of other subcortical structures might also remain in flux throughout life.     

Parvalbumin-expressing interneurons linearly transform cortical responses to visual stimuli

The response of cortical neurons to a sensory stimulus is shaped by the network in which they are embedded. Here we establish a role of parvalbumin (PV)-expressing cells, a large class of inhibitory neurons that target the soma and perisomatic compartments of pyramidal cells, in controlling cortical responses. By bidirectionally manipulating PV cell activity in visual cortex we show that these neurons strongly modulate layer 2/3 pyramidal cell spiking responses to visual stimuli while only modestly affecting their tuning properties. PV cells' impact on pyramidal cells is captured by a linear transformation, both additive and multiplicative, with a threshold. These results indicate that PV cells are ideally suited to modulate cortical gain and establish a causal relationship between a select neuron type and specific computations performed by the cortex during sensory processing.     

5-HT<Subscript>1A</Subscript> receptor expression in pyramidal neurons of cortical and limbic brain regions

We studied expression of the 5-HT_1A receptor in cortical and limbic areas of the brain of the tree shrew. In situ hybridization with a receptor-specific probe and immunocytochemistry with various antibodies was used to identify distinct neurons expressing the receptor. In vitro receptor autoradiography with ³H-8-OH-DPAT (³H-8-hydroxy-2-[di-n-propylamino]tetralin) was performed to visualize receptor-binding sites. In the prefrontal, insular, and occipital cortex, 5-HT_1A receptor mRNA was expressed in pyramidal neurons of layer 2, whereas ³H-8-OH-DPAT labeled layers 1 and 2 generating a columnar-like pattern in the prefrontal and occipital cortex. In the striate and ventral occipital cortex, receptor mRNA was present within layers 5 and 6 in pyramidal neurons and Meynert cells. Pyramid-like neurons in the claustrum and anterior olfactory nucleus also expressed the receptor. Principal neurons in hippocampal region CA1 expressed 5-HT_1A receptor mRNA, and ³H-8-OH-DPAT labeled both the stratum oriens and stratum radiatum. CA3 pyramidal neurons displayed low 5-HT_1A receptor expression, whereas granule neurons in the dentate gyrus revealed moderate expression of this receptor. In the amygdala, large pyramid-like neurons in the basal magnocellular nucleus strongly expressed the receptor. Immunocytochemistry with antibodies against parvalbumin, calbindin, and gamma aminobutyric acid (GABA) provided no evidence for 5-HT_1A receptor expression in GABAergic neurons in cortical and limbic brain areas. Our data agree with previous findings showing that the 5-HT_1A receptor mediates the modulation of glutamatergic neurons. Expression in the limbic and cortical areas suggested an involvement of 5-HT_1A receptors in emotional and cognitive processes.This work was supported by the German Science Foundation (SFB 406; C4 to G.F.).     

Sound-driven synaptic inhibition in primary visual cortex

Multimodal objects and events activate many sensory cortical areas simultaneously. This is possibly reflected in reciprocal modulations of neuronal activity, even at the level of primary cortical areas. However, the synaptic character of these interareal interactions, and their impact on synaptic and behavioral sensory responses are unclear. Here, we found that activation of auditory cortex by a noise burst drove local GABAergic inhibition on supragranular pyramids of the mouse primary visual cortex, via cortico-cortical connections. This inhibition was generated by sound-driven excitation of a limited number of cells in infragranular visual cortical neurons. Consequently, visually driven synaptic and spike responses were reduced upon bimodal stimulation. Also, acoustic stimulation suppressed conditioned behavioral responses to a dim flash, an effect that was prevented by acute blockade of GABAergic transmission in visual cortex. Thus, auditory cortex activation by salient stimuli degrades potentially distracting sensory processing in visual cortex by recruiting local, translaminar, inhibitory circuits.     

Convergence across Tactile Afferent Types in Primary and Secondary Somatosensory Cortices

Integration of information by convergence of inputs onto sensory cortical neurons is a requisite for processing higher-order stimulus features. Convergence across defined peripheral input classes has generally been thought to occur at levels beyond the primary sensory cortex, however recent work has shown that this does not hold for the convergence of slowly-adapting and rapidly-adapting inputs in primary somatosensory cortex. We have used a new analysis method for multi-unit recordings, to show convergence of inputs deriving from the rapidly-adapting and Pacinian channels in a proportion of neurons in both primary and secondary somatosensory cortex in the anaesthetised cat. We have validated this method using single-unit recordings. The secondary somatosensory cortex has a greater proportion of sites that show convergence of this type than primary somatosensory cortex. These findings support the hypothesis that the more complex features processed in higher cortical areas require a greater degree of convergence across input classes, but also shows that this convergence is apparent in the primary somatosensory cortex.     

Sonic hedgehog expression in corticofugal projection neurons directs cortical microcircuit formation

The precise connectivity of inputs and outputs is critical for cerebral cortex function; however, the cellular mechanisms that establish these connections are poorly understood. Here, we show that the secreted molecule Sonic Hedgehog (Shh) is involved in synapse formation of a specific cortical circuit. Shh is expressed in layer V corticofugal projection neurons and the Shh receptor, Brother of CDO (Boc), is expressed in local and callosal projection neurons of layer II/III that synapse onto the subcortical projection neurons. Layer V neurons of mice lacking functional Shh exhibit decreased synapses. Conversely, the loss of functional Boc leads to a reduction in the strength of synaptic connections onto layer Vb, but not layer II/III, pyramidal neurons. These results demonstrate that Shh is expressed in postsynaptic target cells while Boc is expressed in a complementary population of presynaptic input neurons, and they function to guide the formation of cortical microcircuitry. VIDEO ABSTRACT:     

Hippocampus-like corticoneurogenesis induced by two isoforms of the BTB-zinc finger gene Zbtb20 in mice

Hippocampus-associated genes that orchestrate the formation of the compact stratum pyramidale are largely unknown. The BTB (broad complex, tramtrack, bric-a-brac)-zinc finger gene Zbtb20 (also known as HOF, Znf288, Zfp288) encodes two protein isoforms, designated Zbtb20(S) and Zbtb20(L), which are expressed in newborn pyramidal neurons of the presumptive hippocampus in mice. Here, we have generated transgenic mice with ectopic expression of Zbtb20(S) and Zbtb20(L) in immature pyramidal neurons differentiated from multipotent non-hippocampal precursors. The subiculum and posterior retrosplenial areas in these mice were transformed into a three-layered hippocampus-like cortex with a compact homogenous pyramidal cell layer. Severe malformations of lamination occur in neocortical areas, which coincide with a deficiency in expression of cortical lamination markers. The alterations in cortical cytoarchitecture result in behavioral abnormalities suggestive of a deficient processing of visual and spatial memory cues in the cerebral cortex of adult Zbtb20 transgenic mice. Overall, our in vivo data suggest that Zbtb20 functions as a molecular switch for a pathway that induces invariant pyramidal neuron morphogenesis and suppression of cell fate transitions in newborn neurons.     

Odorant receptor expression in the mouse cerebral cortex

Mammalian odorant receptors have been known to be involved not only in odorant detection but also in neuronal development of olfactory sensory neurons. We have examined a possibility of odorant receptor expression in nonolfactory neurons in the mouse. Mouse odorant receptors (M71, C6, and OR3), two of which were already shown to be functionally activated by odorants in heterologous systems, were detected by polymerase chain reactions (PCRs) from the cerebral cortex but not from other brain tissues. Degenerate PCR further suggested that other odorant receptors were also expressed in the mouse cerebral cortex. One of these receptors showed high sequence-match with a putative chick odorant receptor OR7 transiently expressed in the notochord during development. In situ hybridization detected signals for M71 and C6 receptors in the layer II cortical pyramidal neurons located in the occipital pole. In the M71-IRES-tauLacZ mouse, in which M71 expression was genetically marked with tauLacZ, X-gal staining signals were mostly localized in the layer II neurons in the occipital pole, being consistent with the in situ hybridization result. Fluorescent immunohistochemistry using anti-beta-galactosidase antibody further detected the tauLacZ signals in the same cells. X-gal staining began at P3, peaked at P8, and continued to adults, although signals gradually decreased. These data showed that at least a few odorant receptors are expressed not only in olfactory sensory neurons but also in pyramidal neurons in the cerebral cortex, possibly playing an important role either in chemical detection of exogenous or endogenous ligands or in a developmental process such as axon guidance and target recognition.     

Satb1 ablation alters temporal expression of immediate early genes and reduces dendritic spine density during postnatal brain development

Complex behaviors, such as learning and memory, are associated with rapid changes in gene expression of neurons and subsequent formation of new synaptic connections. However, how external signals are processed to drive specific changes in gene expression is largely unknown. We found that the genome organizer protein Satb1 is highly expressed in mature neurons, primarily in the cerebral cortex, dentate hilus, and amygdala. In Satb1-null mice, cortical layer morphology was normal. However, in postnatal Satb1-null cortical pyramidal neurons, we found a substantial decrease in the density of dendritic spines, which play critical roles in synaptic transmission and plasticity. Further, we found that in the cerebral cortex, Satb1 binds to genomic loci of multiple immediate early genes (IEGs) (Fos, Fosb, Egr1, Egr2, Arc, and Bdnf) and other key neuronal genes, many of which have been implicated in synaptic plasticity. Loss of Satb1 resulted in greatly alters timing and expression levels of these IEGs during early postnatal cerebral cortical development and also upon stimulation in cortical organotypic cultures. These data indicate that Satb1 is required for proper temporal dynamics of IEG expression. Based on these findings, we propose that Satb1 plays a critical role in cortical neurons to facilitate neuronal plasticity.     

Hierarchy within human SI: supporting data from cytochrome oxidase,acetylcholinesterase and NADPH-diaphorase staining patterns

The human primary somatosensory cortex consists of four cytoarchitectonic subdivisions (3a, 3b, 1 and 2) that are likely to contain distinct somatosensory representations. The intraareal organization of these areas as well as that of the primary motor cortex (area 4) has been analyzed using histochemical stains of cytochrome oxidase, acetylcholinesterase and NADPH-diaphorase activity in normal human brains. Cytochrome oxidase activity was revealed in individual cortical neurons and neuropil. Areas 4, 3a and 3b were on average darker than areas 1 and 2. The laminar distribution of cytochrome oxidase activity varied in different areas. A prominent dark band was present in layers IV and lower III in areas 3a and 3b and in layer III in areas 1, 2 and 4. Acetylcholinesterase staining revealed fibers and pyramidal cells in layers III and V; stained layer III pyramids were rare in areas 3a and 3b and numerous in areas 1, 2 and 4. NADPH-diaphorase positive elements included Golgi-like stained non-pyramidal neurons and Nissl-like stained pyramidal neurons; the former were found, in small numbers, in layer II of areas 4, 3a, 3b and 1, and the latter in layers III and V of areas 4 and 3a and in layer V of areas 1 and 2. The dark cytochrome oxidase staining of layer IV and the paucity of acetylcholinesterase positive pyramids in areas 3a and 3b resemble the pattern found in primary visual and auditory areas, whereas the dark cytochrome oxidase staining in layer III and abundance of acetylcholinesterase positive pyramids in areas 1 and 2 that of association areas. These results suggest that the four areas included in human SI constitute hierarchical stages of cortical processing, with 3a and 3b corresponding to primary and 1 and 2 to secondary areas.     

Hierarchy within human SI: supporting data from cytochrome oxidase, acetylcholinesterase and NADPH-diaphorase staining patterns

The human primary somatosensory cortex consists of four cytoarchitectonic subdivisions (3a, 3b, 1 and 2) that are likely to contain distinct somatosensory representations. The intraareal organization of these areas as well as that of the primary motor cortex (area 4) has been analyzed using histochemical stains of cytochrome oxidase, acetylcholinesterase and NADPH-diaphorase activity in normal human brains. Cytochrome oxidase activity was revealed in individual cortical neurons and neuropil. Areas 4, 3a and 3b were on average darker than areas 1 and 2. The laminar distribution of cytochrome oxidase activity varied in different areas. A prominent dark band was present in layers IV and lower III in areas 3a and 3b and in layer III in areas 1, 2 and 4. Acetylcholinesterase staining revealed fibers and pyramidal cells in layers III and V; stained layer III pyramids were rare in areas 3a and 3b and numerous in areas 1, 2 and 4. NADPH-diaphorase positive elements included Golgi-like stained non-pyramidal neurons and Nissl-like stained pyramidal neurons; the former were found, in small numbers, in layer II of areas 4, 3a, 3b and 1, and the latter in layers III and V of areas 4 and 3a and in layer V of areas 1 and 2. The dark cytochrome oxidase staining of layer IV and the paucity of acetylcholinesterase positive pyramids in areas 3a and 3b resemble the pattern found in primary visual and auditory areas, whereas the dark cytochrome oxidase staining in layer III and abundance of acetylcholinesterase positive pyramids in areas 1 and 2 that of association areas. These results suggest that the four areas included in human SI constitute hierarchical stages of cortical processing, with 3a and 3b corresponding to primary and 1 and 2 to secondary areas.