Tomographic tract tracing and data driven approaches to unravel complex 3D fiber anatomy of DBS relevant prefrontal projections to the diencephalic-mesencephalic junction in the marmoset.

BACKGROUND: Understanding prefrontal cortex projections to diencephalic-mesencephalic junction (DMJ), especially to subthalamic nucleus (STN) and ventral mesencephalic tegmentum (VMT) helps our comprehension of Deep Brain Stimulation (DBS) in major depression (MD) and obsessive-compulsive disorder (OCD). Fiber routes are complex and tract tracing studies in non-human primate species (NHP) have yielded conflicting results. The superolateral medial forebrain bundle (slMFB) is a promising target for DBS in MD and OCD. It has become a focus of criticism owing to its name and its diffusion weighted-imaging based primary description. OBJECTIVE: To investigate DMJ connectivity in NHP with a special focus on slMFB and the limbic hyperdirect pathway utilizing three-dimensional and data driven techniques. METHODS: We performed left prefrontal adeno-associated virus - tracer based injections in the common marmoset monkey (n = 52). Histology and two-photon microscopy were integrated into a common space. Manual and data driven cluster analyses of DMJ, subthalamic nucleus and VMT together, followed by anterior tract tracing streamline (ATTS) tractography were deployed. RESULTS: Typical pre- and supplementary motor hyperdirect connectivity was confirmed. The advanced tract tracing unraveled the complex connectivity to the DMJ. Limbic prefrontal territories directly projected to the VMT but not STN. DISCUSSION: Intricate results of tract tracing studies warrant the application of advanced three-dimensional analyses to understand complex fiber-anatomical routes. The applied three-dimensional techniques can enhance anatomical understanding also in other regions with complex fiber anatomy. CONCLUSION: Our work confirms slMFB anatomy and enfeebles previous misconceptions. The rigorous NHP approach strengthens the role of the slMFB as a target structure for DBS predominantly in psychiatric indications like MD and OCD.

Anatomical variability, multi-modal coordinate systems, and precision targeting in the marmoset brain.

Localising accurate brain regions needs careful evaluation in each experimental species due to their individual variability. However, the function and connectivity of brain areas is commonly studied using a single-subject cranial landmark-based stereotactic atlas in animal neuroscience. Here, we address this issue in a small primate, the common marmoset, which is increasingly widely used in systems neuroscience. We developed a non-invasive multi-modal neuroimaging-based targeting pipeline, which accounts for intersubject anatomical variability in cranial and cortical landmarks in marmosets. This methodology allowed creation of multi-modal templates (MarmosetRIKEN20) including head CT and brain MR images, embedded in coordinate systems of anterior and posterior commissures (AC-PC) and CIFTI grayordinates. We found that the horizontal plane of the stereotactic coordinate was significantly rotated in pitch relative to the AC-PC coordinate system (10 degrees, frontal downwards), and had a significant bias and uncertainty due to positioning procedures. We also found that many common cranial and brain landmarks (e.g., bregma, intraparietal sulcus) vary in location across subjects and are substantial relative to average marmoset cortical area dimensions. Combining the neuroimaging-based targeting pipeline with robot-guided surgery enabled proof-of-concept targeting of deep brain structures with an accuracy of 0.2 mm. Altogether, our findings demonstrate substantial intersubject variability in marmoset brain and cranial landmarks, implying that subject-specific neuroimaging-based localization is needed for precision targeting in marmosets. The population-based templates and atlases in grayordinates, created for the first time in marmoset monkeys, should help bridging between macroscale and microscale analyses.

Intracellular zinc-dependent TAS2R8 gene expression through CTCF activation.

Taste-2 receptors (TAS2Rs), which belong to the G-protein coupled receptor (GPCR) family, are receptors for bitter taste perception. The aim of this study was to investigate whether zinc deficiency affects the expression of TAS2R genes. The promoter activity of the TAS2R7, TAS2R8, and TAS2R42 genes were determined in Ca9-22 oral squamous cell carcinoma cells cultured in the presence or absence of zinc. Luciferase reporter assays showed that zinc deprivation inhibited TAS2R8 promoter activity, but not the promoter activity of the other two genes. Treatment of the cells with N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), an intracellular chelator of Zn2+, in the presence of 10% fetal bovine serum reduced TAS2R8 promoter activity. Truncation/deletion mutants of TAS2R8 promoter-luciferase constructs showed that the region from nucleotide -1152 to nucleotide -925 was critical for intracellular zinc dependency and contained a CCCTC-binding factor (CTCF) binding motif. A chromatin immunoprecipitation (ChiP) assay showed that CTCF bound specifically to this region, a binding abrogated by zinc deficiency, suggesting that CTCF plays a critical role in zinc-dependent bitter taste perception through TAS2R8.

Axonal Projections From the Middle Temporal Area in the Common Marmoset.

Neural activity in the middle temporal (MT) area is modulated by the direction and speed of motion of visual stimuli. The area is buried in a sulcus in the macaque, but exposed to the cortical surface in the marmoset, making the marmoset an ideal animal model for studying MT function. To better understand the details of the roles of this area in cognition, underlying anatomical connections need to be clarified. Because most anatomical tracing studies in marmosets have used retrograde tracers, the axonal projections remain uncharacterized. In order to examine axonal projections from MT, we utilized adeno-associated viral (AAV) tracers, which work as anterograde tracers by expressing either green or red fluorescent protein in infected neurons. AAV tracers were injected into three sites in MT based on retinotopy maps obtained via in vivo optical intrinsic signal imaging. Brains were sectioned and divided into three series, one for fluorescent image scanning and two for myelin and Nissl substance staining to identify specific brain areas. Overall projection patterns were similar across the injections. MT projected to occipital visual areas V1, V2, V3 (VLP) and V4 (VLA) and surrounding areas in the temporal cortex including MTC (V4T), MST, FST, FSTv (PGa/IPa) and TE3. There were also projections to the dorsal visual pathway, V3A (DA), V6 (DM) and V6A, the intraparietal areas AIP, LIP, MIP, frontal A4ab and the prefrontal cortex, A8aV and A8C. There was a visuotopic relationship with occipital visual areas. In a marmoset in which two tracer injections were made, the projection targets did not overlap in A8aV and AIP, suggesting topographic projections from different parts of MT. Most of these areas are known to send projections back to MT, suggesting that they are reciprocally connected with it.

Single-cell bioluminescence imaging of deep tissue in freely moving animals.

Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.

Silencing of FUS in the common marmoset (Callithrix jacchus) brain via stereotaxic injection of an adeno-associated virus encoding shRNA.

Fused in sarcoma (FUS) is an RNA binding protein that is involved in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). To establish the common marmoset (Callithrix jacchus) as a model for FTLD, we generated a stereotaxic injection-based marmoset model of FUS-silencing. We designed shRNAs against the marmoset FUS gene and generated an AAV9 virus encoding the most effective shRNA against FUS (shFUS). The AAV encoding shFUS (AAV-shFUS) was introduced into the frontal cortex of young adult marmosets, whereas AAV encoding a control shRNA was injected into the contralateral side. We obtained approximately 70-80% silencing of FUS following AAV-shFUS injection. Interestingly, FUS-silencing provoked a proliferation of astrocytes and microglias. Since FTLD is characterized by various emotional deficits, it would be helpful to establish a marmoset model of FUS-silencing in various brain tissues for investigating the pathomechanism of higher cognitive and behavioral dysfunction.

Learning new sequential stepping patterns requires striatal plasticity during the earliest phase of acquisition.

Animals including humans execute motor behavior to reach their goals. For this purpose, they must choose correct strategies according to environmental conditions and shape many parameters of their movements, including their serial order and timing. To investigate the neurobiology underlying such skills, we used a multi-sensor equipped, motor-driven running wheel with adjustable sequences of foothold pegs on which mice ran to obtain water reward. When the peg patterns changed from a familiar pattern to a new pattern, the mice had to learn and implement new locomotor strategies in order to receive reward. We found that the accuracy of stepping and the achievement of water reward improved with the new learning after changes in the peg-pattern, and c-Fos expression levels assayed after the first post-switch session were high in both dorsolateral striatum and motor cortex, relative to post-switch plateau levels. Combined in situ hybridization and immunohistochemistry of striatal sections demonstrated that both enkephalin-positive (indirect pathway) neurons and substance P-positive (direct pathway) neurons were recruited specifically after the pattern switches, as were interneurons expressing neuronal nitric oxide synthase. When we blocked N-methyl-D-aspartate (NMDA) receptors in the dorsolateral striatum by injecting the NMDA receptor antagonist, D-2-amino-5-phosphonopentanoic acid (AP5), we found delays in early post-switch improvement in performance. These findings suggest that the dorsolateral striatum is activated on detecting shifts in environment to adapt motor behavior to the new context via NMDA-dependent plasticity, and that this plasticity may underlie forming and breaking skills and habits as well as to behavioral difficulties in clinical disorders.

Application of viral vectors to the study of neural connectivities and neural circuits in the marmoset brain.

It is important to study the neural connectivities and functions in primates. For this purpose, it is critical to be able to transfer genes to certain neurons in the primate brain so that we can image the neuronal signals and analyze the function of the transferred gene. Toward this end, our team has been developing gene transfer systems using viral vectors. In this review, we summarize our current achievements as follows. 1) We compared the features of gene transfer using five different AAV serotypes in combination with three different promoters, namely, CMV, mouse CaMKII (CaMKII), and human synapsin 1 (hSyn1), in the marmoset cortex with those in the mouse and macaque cortices. 2) We used target-specific double-infection techniques in combination with TET-ON and TET-OFF using lentiviral retrograde vectors for enhanced visualization of neural connections. 3) We used an AAV-mediated gene transfer method to study the transcriptional control for amplifying fluorescent signals using the TET/TRE system in the primate neocortex. We also established systems for shRNA mediated gene targeting in a neocortical region where a gene is significantly expressed and for expressing the gene using the CMV promoter for an unexpressed neocortical area in the primate cortex using AAV vectors to understand the regulation of downstream genes. Our findings have demonstrated the feasibility of using viral vector mediated gene transfer systems for the study of primate cortical circuits using the marmoset as an animal model. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 354-372, 2017.

Long-Term Two-Photon Calcium Imaging of Neuronal Populations with Subcellular Resolution in Adult Non-human Primates.

Two-photon imaging with genetically encoded calcium indicators (GECIs) enables long-term observation of neuronal activity in vivo. However, there are very few studies of GECIs in primates. Here, we report a method for long-term imaging of a GECI, GCaMP6f, expressed from adeno-associated virus vectors in cortical neurons of the adult common marmoset (Callithrix jacchus), a small New World primate. We used a tetracycline-inducible expression system to robustly amplify neuronal GCaMP6f expression and up- and downregulate it for more than 100 days. We succeeded in monitoring spontaneous activity not only from hundreds of neurons three-dimensionally distributed in layers 2 and 3 but also from single dendrites and axons in layer 1. Furthermore, we detected selective activities from somata, dendrites, and axons in the somatosensory cortex responding to specific tactile stimuli. Our results provide a way to investigate the organization and plasticity of cortical microcircuits at subcellular resolution in non-human primates.

In Vivo Two-Photon Imaging of Dendritic Spines in Marmoset Neocortex

Two-photon microscopy in combination with a technique involving the artificial expression of fluorescent protein has enabled the direct observation of dendritic spines in living brains. However, the application of this method to primate brains has been hindered by the lack of appropriate labeling techniques for visualizing dendritic spines. Here, we developed an adeno-associated virus vector-based fluorescent protein expression system for visualizing dendritic spines in vivo in the marmoset neocortex. For the clear visualization of each spine, the expression of reporter fluorescent protein should be both sparse and strong. To fulfill these requirements, we amplified fluorescent signals using the tetracycline transactivator (tTA)-tetracycline-responsive element system and by titrating down the amount of Thy1S promoter-driven tTA for sparse expression. By this method, we were able to visualize dendritic spines in the marmoset cortex by two-photon microscopy in vivo and analyze the turnover of spines in the prefrontal cortex. Our results demonstrated that short spines in the marmoset cortex tend to change more frequently than long spines. The comparison of in vivo samples with fixed samples showed that we did not detect all existing spines by our method. Although we found glial cell proliferation, the damage of tissues caused by window construction was relatively small, judging from the comparison of spine length between samples with or without window construction. Our new labeling technique for two-photon imaging to visualize in vivo dendritic spines of the marmoset neocortex can be applicable to examining circuit reorganization and synaptic plasticity in primates.

Comparative analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral cortex.

Here we investigated the transduction characteristics of adeno-associated viral vector (AAV) serotypes 1, 2, 5, 8 and 9 in the marmoset cerebral cortex. Using three constructs that each has hrGFP under ubiquitous (CMV), or neuron-specific (CaMKII and Synapsin I (SynI)) promoters, we investigated (1) the extent of viral spread, (2) cell type tropism, and (3) neuronal transduction efficiency of each serotype. AAV2 was clearly distinct from other serotypes in small spreading and neuronal tropism. We did not observe significant differences in viral spread among other serotypes. Regarding the cell tropism, AAV1, 5, 8 and 9 exhibited mostly glial expression for CMV construct. However, when the CaMKII construct was tested, cortical neurons were efficiently transduced (>∼70% in layer 3) by all serotypes, suggesting that glial expression obscured neuronal expression for CMV construct. For both SynI and CaMKII constructs, we observed generally high-level expression in large pyramidal cells especially in layer 5, as well as in parvalbumin-positive interneurons. The expression from the CaMKII construct was more uniformly observed in excitatory cells compared with SynI construct. Injection of the same viral preparations in mouse and macaque cortex resulted in essentially the same result with some species-specific differences.

Simultaneous visualization of extrinsic and intrinsic axon collaterals in Golgi-like detail for mouse corticothalamic and corticocortical cells: a double viral infection method.

Here we present a novel tracing technique to stain projection neurons in Golgi-like detail by double viral infection. We used retrograde lentiviral vectors and adeno-associated viral vectors (AAV) to drive "TET-ON/TET-OFF system" in neurons connecting two regions. Using this method, we successfully labeled the corticothalamic (CT) cells of the mouse somatosensory barrel field (S1BF) and motor cortex (M1) in their entirety. We also labeled contra- and ipsilaterally-projecting corticocortical (CC) cells of M1 by targeting contralateral M1 or ipsilateral S1 for retrograde infection. The strength of this method is that we can observe the morphology of specific projection neuron subtypes en masse. We found that the group of CT cells extends their dendrites and intrinsic axons extensively below but not within the thalamorecipient layer in both S1BF and M1, suggesting that the primary target of this cell type is not layer 4. We also found that both ipsi- and contralateral targeting CC cells in M1 commonly exhibit widespread collateral extensions to contralateral M1 (layers 1-6), bilateral S1 and S2 (layers 1, 5 and 6), perirhinal cortex (layers 1, 2/3, 5, and 6), striatum and claustrum. These findings not only strengthened the previous findings of single cell tracings but also extended them by enabling cross-area comparison of CT cells or comparison of CC cells of two different labeling.

Genetic dissection of the circuit for hand dexterity in primates.

It is generally accepted that the direct connection from the motor cortex to spinal motor neurons is responsible for dexterous hand movements in primates. However, the role of the 'phylogenetically older' indirect pathways from the motor cortex to motor neurons, mediated by spinal interneurons, remains elusive. Here we used a novel double-infection technique to interrupt the transmission through the propriospinal neurons (PNs), which act as a relay of the indirect pathway in macaque monkeys (Macaca fuscata and Macaca mulatta). The PNs were double infected by injection of a highly efficient retrograde gene-transfer vector into their target area and subsequent injection of adeno-associated viral vector at the location of cell somata. This method enabled reversible expression of green fluorescent protein (GFP)-tagged tetanus neurotoxin, thereby permitting the selective and temporal blockade of the motor cortex­PN­motor neuron pathway. This treatment impaired reach and grasp movements, revealing a critical role for the PN-mediated pathway in the control of hand dexterity. Anti-GFP immunohistochemistry visualized the cell bodies and axonal trajectories of the blocked PNs, which confirmed their anatomical connection to motor neurons. This pathway-selective and reversible technique for blocking neural transmission does not depend on cell-specific promoters or transgenic techniques, and is a new and powerful tool for functional dissection in system-level neuroscience studies.

Altered layer-specific gene expression in cortical samples from patients with temporal lobe epilepsy.

PURPOSE: Neuropathologic investigations frequently reveal the presence of architectural cortical dysplasia in patients with temporal lobe epilepsy (TLE), sometimes as an isolated finding but more commonly associated with hippocampal sclerosis (HS) and white matter abnormalities. The histologic pattern and the developmental origin of these alterations are not clear, and their diagnostic criteria are poorly defined. The aim of this study was to investigate the expression patterns of layer-specific genes in cortical specimens from patients with TLE presenting different subtypes of cortical malformations in order to elucidate the disorganization of the laminar architecture of such epileptogenic abnormalities and provide evidence to enable a more objective neuropathologic diagnosis. METHODS: We analyzed the expression patterns of CUX2, RORBETA, ER81, NURR1, and CTGF genes, respectively specific markers of layers II-III, IV, V, VI, and VIb, in surgical samples by means of in situ hybridization and compared them with those observed in control cortices. The pathologic samples included typical architectural dysplasia (group 1); temporal lobe sclerosis, a variant of architectural dysplasia (group 2); and white matter heterotopic neuronal aggregates, namely small lentiform nodules (group 3). These abnormalities may have been associated or not with HS. KEY FINDINGS: All of the genes had a laminar expression pattern in normal cortices, whereas groups 1 and 2 showed alterations mainly involving layers V and VI, and highlighted by the altered distribution of ER81- and NURR1-positive cells. The expression of ER81 and NURR1 genes was different among the groups, and atypical coexpression of NURR1 and CUX2 mRNA was detected in the neurons making up the small lentiform nodules. SIGNIFICANCE: These findings indicate that defects in cortical organization involving the deeper cortical neurons may be a common etiopathogenic mechanism in group 1 and 2 cortical dysplasia, whether isolated or associated with HS, and that developmental disorders may also be present in the white matter (group 3). They also provide evidence that the layer-specific genes can be usefully used to investigate the neuropathology of human cortical dysplasia.

Expression of ErbB4 in substantia nigra dopamine neurons of monkeys and humans.

Abnormal neuregulin-1 signaling through its receptor (ErbB4) might be associated with schizophrenia, although their neuropathological contribution remains controversial. To assess the role of neuregulin-1 in the dopamine hypothesis of schizophrenia, we used in situ hybridization and immunoblotting to investigate the cellular distribution of ErbB4 mRNA in the substantia nigra of Japanese monkeys (Macaca fuscata) and human postmortem brains. In both monkeys and humans, significant signal for ErbB4 mRNA was detected in substantia nigra dopamine neurons, which were identified by melanin deposits. The expression of ErbB4 mRNA in nigral dopamine neurons was confirmed with an independent RNA probe, as well as with combined tyrosine hydroxylase immunostaining. Immunoblotting appeared to support the observation of in situ hybridization. Immunoreactivity for ErbB4 protein was much more enriched in substantia nigra pars compacta containing dopamine neurons than in neighboring substantia nigra pars reticulata. These observations suggest that ErbB4 is expressed in the dopaminergic neurons of primate substantia nigra and ErbB4 abnormality might contribute to the dopaminergic pathology associated with schizophrenia or other brain diseases.

Paraneoplastic antigen-like 5 gene (PNMA5) is preferentially expressed in the association areas in a primate specific manner.

To understand the relationship between the structure and function of primate neocortical areas at a molecular level, we have been screening for genes differentially expressed across macaque neocortical areas by restriction landmark cDNA scanning (RLCS). Here, we report enriched expression of the paraneoplastic antigen-like 5 gene (PNMA5) in association areas but not in primary sensory areas, with the lowest expression level in primary visual cortex. In situ hybridization in the primary sensory areas revealed PNMA5 mRNA expression restricted to layer II. Along the ventral visual pathway, the expression gradually increased in the excitatory neurons from the primary to higher visual areas. This differential expression pattern was very similar to that of retinol-binding protein (RBP) mRNA, another association-area-enriched gene that we reported previously. Additional expression analysis for comparison of other genes in the PNMA gene family, PNMA1, PNMA2, PNMA3, and MOAP1 (PNMA4), showed that they were widely expressed across areas and layers but without the differentiated pattern of PNMA5. In mouse brains, PNMA1 was only faintly expressed and PNMA5 was not detected. Sequence analysis showed divergence of PNMA5 sequences among mammals. These findings suggest that PNMA5 acquired a certain specialized role in the association areas of the neocortex during primate evolution.

Differential expression patterns of occ1-related genes in adult monkey visual cortex.

We have previously revealed that occ1 is preferentially expressed in the primary visual area (V1) of the monkey neocortex. In our attempt to identify more area-selective genes in the macaque neocortex, we found that testican-1, an occ1-related gene, and its family members also exhibit characteristic expression patterns along the visual pathway. The expression levels of testican-1 and testican-2 mRNAs as well as that of occ1 mRNA start of high in V1, progressively decrease along the ventral visual pathway, and end of low in the temporal areas. Complementary to them, the neuronal expression of SPARC mRNA is abundant in the association areas and scarce in V1. Whereas occ1, testican-1, and testican-2 mRNAs are preferentially distributed in thalamorecipient layers including "blobs," SPARC mRNA expression avoids these layers. Neither SC1 nor testican-3 mRNA expression is selective to particular areas, but SC1 mRNA is abundantly observed in blobs. The expressions of occ1, testican-1, testican-2, and SC1 mRNA were downregulated after monocular tetrodotoxin injection. These results resonate with previous works on chemical and functional gradients along the primate occipitotemporal visual pathway and raise the possibility that these gradients and functional architecture may be related to the visual activity-dependent expression of these extracellular matrix glycoproteins.

Activation of syntaxin 1C, an alternative splice variant of HPC-1/syntaxin 1A, by phorbol 12-myristate 13-acetate (PMA) suppresses glucose transport into astroglioma cells via the glucose transporter-1 (GLUT-1).

Syntaxin 1C is an alternative splice variant lacking the transmembrane domain of HPC-1/syntaxin 1A. We found previously that syntaxin 1C is expressed as a soluble protein in human astroglioma (T98G) cells, and syntaxin 1C expression is enhanced by stimulation with phorbol 12-myristate 13-acetate (PMA). However, the physiological function of syntaxin 1C is not known. In this study, we examined the relationship between syntaxin 1C and glucose transport. First, we discovered that glucose transporter-1 (GLUT-1) was the primary isoform in T98G cells. Second, we demonstrated that glucose uptake in T98G cells was suppressed following an increase in endogenous syntaxin 1C after stimulation with PMA, which did not alter the expression levels of other plasma membrane syntaxins. We further examined glucose uptake and intracellular localization of GLUT-1 in cells that overexpressed exogenous syntaxin 1C; glucose uptake via GLUT-1 was inhibited without affecting sodium-dependent glucose transport. The value of Vmax for the dose-dependent uptake of glucose was reduced in syntaxin 1C-expressing cells, whereas there was no change in Km. Immunofluorescence studies revealed a reduction in the amount of GLUT-1 in the plasma membrane in cells that expressed syntaxin 1C. Based on these results, we postulate that syntaxin 1C regulates glucose transport in astroglioma cells by changing the intracellular trafficking of GLUT-1. This is the first report to indicate that a syntaxin isoform that lacks a transmembrane domain can regulate the intracellular transport of a plasma membrane protein.

Ciliary neurotrophic factor inhibits differentiation of photoreceptor-like cells in rat pineal glands in vitro.

Ciliary neurotrophic factor (CNTF) is a unique member of the interleukin-6 (IL-6) family, whose receptor subunit for ligand binding is exclusively expressed in the nervous system and muscle. The role of CNTF in mammalian development remains unknown. We recently reported the specific expression of CNTF in the pineal gland and eyes. To further examine the expression pattern and role of CNTF in development, we prepared a polyclonal antibody against rat CNTF, performed western blotting with this antibody, and confirmed a strong and specific expression of the CNTF protein in pineal glands and a moderate expression in the eyes among the various tissues examined in newborn rats. In pineal organ cultures of newborn rats, exogenously added recombinant rat CNTF potently inhibited the differentiation of photoreceptor-like cells in a dose-dependent manner, while CNTF did not influence the survival of pineal cells. Among several cell growth factors known to have a similar effect in retinal cultures examined, strong inhibitory effects were seen only with CNTF and the leukemia inhibitory factor (LIF), both of which belong to the IL-6 cytokine family. This inhibitory effect was the strongest during three to 6 days of culture when CNTF was added to these cultures. These results suggest that CNTF plays an inhibitory role in the development of photoreceptor-like cells in early postnatal rat pineal glands.