I-type Cells Research Articles

Abstract 5216: Atypical PKC inhibitors ICA-1S and ζ-Stat show inhibition of neuroblastoma cell proliferation

Abstract Neuroblastoma, the pediatric cancer of the early nerve cells, has the tendency to become resistant to traditional therapeutics due to its highly heterogeneous nature. Atypical Protein Kinase C-iota (PKC- ι) has been found to promote cell proliferation in neuroblastoma cells through the PKC- ι /Cdk7/Cdk2 signaling pathway. In this study we utilized various cell lines; I-type (intermediate) CHP-212& BE(2)-C, N-types (neuroblastic); BE(2)-M17& SH-SY5Y and S-types (substrate adherent); SK-N-BE(2) & LAI-5s, representing neuroblastoma heterogeneity to investigate the use of aPKC inhibitors as potential neuroblastoma therapeutics. The aPKC inhibitors, 5-amino-1-2,3-dihydroxy-4-(methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) (PKC- ι specific) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) (PKC- ζ specific), both display the ability to reduce neuroblastoma proliferation in a cell type specific manner. ICA-1S was shown to reduce proliferation of BE(2)-C, BE(2)-M17, LAI-5s and SK-N-BE(2) to varying degrees. ζ-Stat was able to consistently reduce proliferation of BE(2)-C, BE(2)-M17, SK-N-BE(2), LAI-5s. The degree of proliferation inhibition varied by drug and cell line. Increasing concentration of ICA-1S displayed an inverse relationship between increasing concentration and a decrease in cell proliferation in the four cell lines previously mentioned. ζ-Stat showed a similar relationship to ICA-1S, however had a lower level inhibition than ICA-1S. WST-1 analysis showed that both drugs displayed varying degrees of toxicity in a cell line dependent manner. The results show that I-type cell lines showed the most linearly proportional toxicity to ICA-1S and ζ-Stat in that increasing concentration increased drug toxicity. N-type cell lines displayed a disproportional relationship between concentration and toxicity. The S-type cell lines followed suit with the N-type in their varying degrees of toxicity. Western blot analysis shows that both inhibitors were effective in reducing the expression of total aPKC levels. We observed the downregulation of various mesenchymal markers using the inhibitors. In particular, ICA-1S appears to have a high percentage of decreased expression of N-cadherin. We also accessed the degree of phosphorylation of Vimentin, S33, S39 and S56 positions which are crucial for its activation. In summary both inhibitors prove to be promising potential neuroblastoma therapeutics. The drugs appear to behave in a more cytostatic manner inhibiting the proliferation of neuroblastoma cells. Further western blots ae still ongoing to determine other pathways through which the drugs act to decrease proliferation. Ongoing experiments include the analysis of tumors collected from mouse models to ascertain the effects of the drugs in vivo as well as the analysis of human tumor samples to ascertain the expression of aPKCs and other markers in patient populations. Experiments also include the use of gene expression knockdowns to compare with the effects from the inhibitors as well as the use of microscopy techniques; fluorescent and electron miscopy to observe the localization of mesenchymal and proliferative markers. Citation Format: Sloan Breedy, Wishrawana S. Ratnayake, Christopher Apostolatos, Avijit Dey, Mildred Acevedo-Duncan. Atypical PKC inhibitors ICA-1S and ζ-Stat show inhibition of neuroblastoma cell proliferation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5216.

Differentiation-Induced Remodelling of Store-Operated Calcium Entry Is Independent of Neuronal or Glial Phenotype but Modulated by Cellular Context

Neurogenesis is a complex process leading to the generation of neuronal networks and glial cell types from stem cells or intermediate progenitors. Mapping subcellular and molecular changes accompanying the switch from proliferation to differentiation is vital for developing therapeutic targets for neurological diseases. Neuronal (N-type) and glial (S-type) phenotypes within the SH-SY5Y neuroblastoma cell line have distinct differentiation responses to 9-cis-retinoic acid (9cRA). In both cell phenotypes, these were accompanied at the single cell level by an uncoupling of Ca2+ store release from store-operated Ca2+ entry (SOCE), mediated by changes in the expression of calcium release-activated calcium pore proteins. This remodelling of calcium signalling was moderated by the predominant cell phenotype within the population. N- and S-type cells differed markedly in their phenotypic stability after withdrawal of the differentiation inducer, with the phenotypic stability of S-type cells, both morphologically and with respect to SOCE properties, in marked contrast to the lability of the N-type phenotype. Furthermore, the SOCE response of I-type cells, a presumed precursor to both N- and S-type cells, varied markedly in different cell environments. These results demonstrate the unique biology of neuronal and glial derivatives of common precursors and suggest that direct or indirect interactions between cell types are vital components of neurogenesis that need to be considered in experimental models.

A distinct gene expression signature characterizes human neuroblastoma cancer stem cells

Neuroblastoma, a malignancy of multipotent embryonic neural crest cells, is the most common extracranial solid cancer in childhood and most common cancer in infancy. Cellular phenotype has been shown to be an important determinant of the malignant potential in human neuroblastoma cells and tumors. Whereas neuroblastic (N-type) are moderately malignant and nonneuronal (S-type) cells are nonmalignant, I-type stem cells are highly tumorigenic, irrespective of N-myc amplification status. In the present study, we sought to determine which genes were overexpressed in the I-type cells which might characterize and maintain the stem cell state and/or malignancy of human neuroblastoma cancer stem cells. We used a microarray platform to compare the steady-state expression levels of mRNAs from 13 human neuroblastoma cell lines representing the three cellular phenotypes. Using qRT-PCR and Western blot analyses, we identified seven genes whose expression is consistently elevated exclusively in neuroblastoma cancer stem cells: CD133, KIT, NOTCH1, GPRC5C, PIGF2, TRKB, and LNGFR. Moreover, we show that the genes are phenotype specific, as differentiation of I-type BE(2)-C cells to either an N- or S-type morphology results in significantly reduced mRNA expression. Finally, we show that NOTCH1 plays an important role in maintaining the stem cell phenotype. The identification and characterization of these genes, elevated in highly malignant neuroblastoma stem cells, could provide the basis for developing novel therapies for treatment of this lethal childhood cancer.

Motion processing across multiple topographic maps in the electrosensory system.

Animals can efficiently process sensory stimuli whose attributes vary over orders of magnitude by devoting specific neural pathways to process specific features in parallel. Weakly electric fish offer an attractive model system as electrosensory pyramidal neurons responding to amplitude modulations of their self‐generated electric field are organized into three parallel maps of the body surface. While previous studies have shown that these fish use parallel pathways to process stationary stimuli, whether a similar strategy is used to process motion stimuli remains unknown to this day. We recorded from electrosensory pyramidal neurons in the weakly electric fish Apteronotus leptorhynchus across parallel maps of the body surface (centromedial, centrolateral, and lateral) in response to objects moving at velocities spanning the natural range. Contrary to previous observations made with stationary stimuli, we found that all cells responded in a similar fashion to moving objects. Indeed, all cells showed a stronger directionally nonselective response when the object moved at a larger velocity. In order to explain these results, we built a mathematical model incorporating the known antagonistic center–surround receptive field organization of these neurons. We found that this simple model could quantitatively account for our experimentally observed differences seen across E and I‐type cells across all three maps. Our results thus provide strong evidence against the hypothesis that weakly electric fish use parallel neural pathways to process motion stimuli and we discuss their implications for sensory processing in general.

Downregulation of OCT4 promotes differentiation and inhibits growth of BE (2)-C human neuroblastoma I-type cells

In invitro continuous culture, N (neuroblastic)-, S(substrate adherent)- and I (intermediate)-type cells were identified in human neuroblastoma (NB), where I-type is recognized as a stem cell type. Octamer-binding protein 4 (OCT4) is a cell marker used to identify the stemness of cells, whose roles in regulating I-type NB cells have yet to be fully elucidated. In the present study, we assessed the differentiation regulation role of OCT4 in BE (2)-C of I-type cells. We demonstrated that downregulation of OCT4 expression in BE (2)-C was associated with reduced cell proliferation and loss of colony formation ability on soft agar, and prompted BE (2)-C cells to differentiate to S-type cells. By contrast, overexpression of OCT4 increased cell proliferation and colony formation ratio, but no obvious differentiation promotion was observed. Furthermore, induced differentiation of BE (2)-C cells to S-type by 5-bromo-2'-deoxyuridine (BrdUrd) was accompanied by reduced expression of OCT4.

Store-operated Ca2 + entry in proliferating and retinoic acid-differentiated N- and S-type neuroblastoma cells

Neuroblastoma cell lines are heterogeneous, comprised of at least three distinct cell phenotypes; neuroblastic N-type cells, non-neuronal substrate-adherent S-type cells and intermediate I-type cells. N- and S-type cell populations were enriched from the parental SH-SY5Y neuroblastoma cell line and induced to differentiate by the addition of retinoic acid (RA), a drug used in the treatment of neuroblastoma. N- and S-type cells were identified based on their differential expression of β-tubulin III, vimentin and Bcl-2. Store-operated Ca2+ entry (SOCE) was then measured in proliferating and differentiated N- and S-type cell populations and the expression of STIM1, Orai1 and TRPC1, three proteins reported to play a key role in SOCE, was determined. In N-type cells the RA-induced switch from proliferation to differentiation was accompanied by a down-regulation in SOCE. STIM1 and Orai1 expression became down-regulated in differentiated cells, consistent with their respective roles as ER Ca2+ sensor and store-operated Ca2+ channel (SOC). TRPC1 became up-regulated suggesting that TRPC1 is not involved in SOCE, at least in differentiated N-type cells. In S-type cells SOCE remained active following the RA-induced switch from proliferation to differentiation and the expression of STIM1 and Orai1 remained unchanged. TRPC1 was not expressed in S-type cells. Our results indicate that differentiation of neuronal cells is associated with a remodelling of SOCE. Therapeutic targeting of SOCE proteins could potentially be a means of promoting neuronal differentiation in the treatment of neuroblastoma.

Comprehensive characterization of neuroblastoma cell line subtypes reveals bilineage potential similar to neural crest stem cells

BackgroundNeuroblastic tumors (NBT) derive from neural crest stem cells (NCSC). Histologically, NBT are composed by neuroblasts and Schwannian cells. In culture, neuroblastic (N-), substrate-adherent (S-) and intermediate phenotype (I-) cell subtypes arise spontaneously.MethodsHere, neuroblastoma (NB) cell line subtypes were characterized according to embryonic peripheral nervous system development markers (GAP43, Phox2b, Sox10, c-kit, GD2, NF68, vimentin, S100β, calcyclin and ABCG2), morphological features, gene expression and differentiation potential. I-type cells were investigated as a bipotential (neuronal and glial) differentiation stage.ResultsPositive immunostaining of NCSC (GAP43, c-kit, NF68, vimentin and Phox2b) and undifferentiated cell (ABCG2) markers was observed in all NB subtypes. N- and I-type cells displayed cytoplasmic membrane GD2 staining, while nuclear calcyclin was restricted to S-type. N- and I-type cells showed similar phenotype and immunoreactivity pattern. Differential gene expression was associated with each cell subtype. N- and I-type cells displayed similar differentiation capacity towards neuronal and glial lineage fates. S-type cells, upon induction, did not show a neuronal-like phenotype, despite gene expression changes.ConclusionResults suggest that N- and I-type NB cell subtypes represent an immature bilineage stage, able to progress towards neuronal and glial fates upon induction of differentiation. S-type cells appear irreversibly committed to a glial lineage fate.

Latent membrane protein-1 of Epstein-Barr virus induces the expression of B-cell integration cluster, a precursor form of microRNA-155, in B lymphoma cell lines

miR-155, a microRNA, and its precursor form, B-cell integration cluster (BIC), are involved in tumor growth. Epstein-Barr virus (EBV)-associated malignancies are categorized into three types, based on their latent gene expression pattern: latency I, II, and III. In the present study, we found that infection with EBV increased the expression of BIC; in addition, substantial expression of BIC/miR-155 was detected in latency III-, but not in latency I-type cells. In comparison, latent membrane protein-1 (LMP1) was expressed in latency III-type cells. When LMP1 was over-expressed, BIC expression increased, indicating LMP1 mediates BIC expression. LMP1 is a membrane-associated protein known to activate signaling pathways. With the use of pathway inhibitors, we found that LMP1-induced strong BIC expression, primarily through NF-κB and p38/MAPK pathways. These results suggest that BIC/miR-155 play a role in lymphomagenesis through NF-κB and p38/MAPK pathways in response to activation by EBV LMP1.

Bmi-1 Regulates the Differentiation and Clonogenic Self-renewal of I-type Neuroblastoma Cells in a Concentration-dependent Manner

Human neuroblastoma I-type cells have been proposed as a population of malignant neural crest stem cells, based on their high tumorigenic potential, expression of stem cell markers, and ability to differentiate into cells of neural crest lineages, including neuroblastic (N-type) and Schwann/glial (S-type) cells. Here, we demonstrate at single cell levels that a subpopulation of I-type cells possess clonogenic self-renewal capacity that requires the Polycomb group family transcription repressor Bmi-1. We further show that Bmi-1 expression levels exert an instructive influence on lineage commitment by I-type cells. Spontaneous and induced differentiation of I-type cells into S-type cells is accompanied by a marked reduction in the level of Bmi-1 expression, and enforced down-regulation of BMI-1 facilitates spontaneous differentiation of I-type cells into S-type cells. By contrast, N-type neuroblastoma cell lines and differentiated N-type cells express higher levels of Bmi-1 relative to I-type cells, and overexpression of BMI-1 promotes the differentiation of I-type cells along the neuronal lineage. Thus, Bmi-1 acts in a concentration-dependent manner in the control of the delicate balance between the self-renewal and differentiation of neuroblastoma I-type cells. These observations suggest that graded activation of a master regulator within individual tumors could trigger divergent developmental programs responsible for both tumor growth and heterogeneity.

Characteristics of Stem Cells from Human Neuroblastoma Cell Lines and in Tumors

Cellular heterogeneity is a hallmark of human neuroblastoma tumors and cell lines. Within a single neuroblastoma are cells from distinct neural crest lineages whose relative abundance is significant for prognosis. We postulate that a self-renewing multipotent tumor stem cell, which gives rise to diverse cell lineages, is the malignant progenitor of this cancer. To test this hypothesis, we have established 22 cloned, phenotypically homogeneous populations of the three major cell types from 17 neuroblastoma cell lines. In vitro, malignant neuroblastoma stem cells, termed 1-type (intermediate type), have distinct morphologic, biochemical, differentiative, and tumorigenic properties. I-type cells express features of both neuroblastic (N) cells (scant cytoplasm, neuritic processes, neurofilaments, pseudoganglia, and granin and neurotransmitter enzyme expression) and substrate-adherent (S) cells (extensive cytoplasm and vimentin and CD44 expression). Moreover, they show bidirectional differentiation to either N or S cells when induced by specific agents. I-type cells are significantly more malignant than N- or S-type cells, with four- to five-fold greater plating efficiencies in soft agar and six-fold higher tumorigenicity in athymic mice. Differences in malignant potential are unrelated to N-myc amplification/overexpression or the ability to digest and migrate through the extracellular matrix. Immunocytochemical analyses of a small series of tumors reveal that frequency of cells coexpressing N and S cell markers correlates with poor prognosis. Thus, I-type stem cells may be instrumental in the genesis and growth of tumors in the patient. Their unique biology deserves attention and further investigation.

A role for distinct cell types in determining malignancy in human neuroblastoma cell lines and tumors

Human neuroblastoma arises from the developing neural crest. Tumors are categorized clinically by their location, age at diagnosis, spread/metastasis, and degree of cellular maturation and heterogeneity. Our long-term studies have shown the presence in human neuroblastoma cell lines of three distinct cell types: I-type stem cells, N-type neuroblastic/neuroendocrine precursors, and S-type Schwannian/melanoblastic precursors. These distinct cell types can differentiate predictably along specific neural crest lineages in response to particular morphogens. As assessed by tumor formation in nude mice and anchorage-independent growth in soft agar, I-type stem cells are significantly more malignant than either N- or S-type cells. Recent research shows that three similar cell types are also present in human neuroblastoma tumors. Using immunocytochemical, laser-capture microdissection, or short-term culture methods to identify cell types in tumors of different stages and/or different outcomes, these studies have shown that (1) all tumors contain neuroblasts in various differentiation states; (2) presumptive I-type stem cells are present in tumors of all stages; and (3) stromal cells may be tumor-derived, i.e. S-type cells, as well as of normal origin. More importantly, there is a higher incidence of I-type cells in tumors that progress, consistent with the high malignant potential of this cell type in vitro. A better understanding of the cause and consequences of cellular heterogeneity of human neuroblastoma tumors is an important prerequisite to the development of more effective therapies for this often fatal disease.

Synthesis and Crystal Structure Determination of Tl8Nb27.2O72Using TEM and Single-Crystal X-Ray Diffraction

An investigation of the Nb–Tl–O system has allowed the synthesis of a new phase, Tl8Nb27.2O72. This phase is structurally and chemically close to a Tl–Nb–O phase previously described as a builtup hexagonal bronze witha≈7.4 Å,b≈13 Å,c≈7.6 Å, and an orthorhombic C-type lattice. The electron diffraction study evidences a doubling of thecparameter with regard to the C cell, resulting in an orthorhombic I-type lattice with, in addition, the observation of a second superstructure and twinned microdomains. A structural determination of the mean orthorhombic I-type cell was successfully carried out by combining energy dispersion spectroscopy (EDS) and single-crystal XRD studies and leads toa=7.534(7) Å,b=12.992(12) Å,c=15.555(12) Å,Z=8, space groupIm2m,R=0.097, andRw=0.099 from 995 independent reflections. A chemical formula for this thallium niobate, Tl8Nb27.2O72, consistent with EDS results, was deduced by structure refinement. HREM investigations on this phase clearly show irregular occupancy of the supplementary crystallographic positions by niobium atoms together with a monocommensurate modulated structure and twinned microdomains.

A cDNA encoding a putative 37 kDa leucine-rich repeat (LRR) protein, p37NB, isolated from S-type neuroblastoma cell has a differential tissue distribution

In human neuroblastoma cells in culture, three morphologically distinct types of cells are observed: neuroblastic N-type cells, Schwannian S-type cells, and intermediate I-type cells. To investigate the differences in gene expression between N-type LA1-55N and S-type LA1-5S cells of the human neuroblastoma cell line LA-N-1, we constructed a subtractive cDNA library from LA1-5S cells. One of the genes that are expressed more in S-type cells than in N-type cells was identified as previously undescribed and is the ocus of this report. We cloned a full-length cDNA of this gene, p37NB, and determined its sequence. A homology search against the GenBank database showed that this was from a novel gene encoding a putative 37 kDa leucine-rich repeat (LRR) protein. Northern blot hybridization and RT-PCR showed that the p37NB gene was differentially expressed in S-type compared to N-type cells of a few neuroblastoma cell lines.

Expression and distribution of peripherin protein in human neuroblastoma cell lines

A series of human neuroblastoma (NB) cell lines was analyzed for expression of peripherin, a class-III intermediate filament protein expressed at high levels in ganglia of the peripheral nervous system. By Western blotting, peripherin protein was detected in all human NB cell lines examined. The highest level of peripherin was found in the NUB-7 cell line, previously characterized as homogeneously neuroblastic. By immunofluorescence labeling, peripherin was shown to be organized in a perinuclear filamentous pattern and, exemplified by IMR32 cells, was also shown to be localized to spontaneously formed neurites. Peripherin was expressed in neuroblastic but not substrate-adherent cells, and was found at low levels in I-type cells. There was a pronounced redistribution of peripherin to neurites formed in response to dibutyryl cyclic adenosine monophosphate (dbcAMP) and all-trans-retinoic acid (RA). In NUB-7 cells, which do not extend neurites in response to nerve growth factor, there was no change in the level of peripherin protein following treatment with this agent. Both dbcAMP and RA induced a redistribution of peripherin to neurite extensions, but only treatment with RA increased the level of the protein as demonstrated with NUB-6A4 and NUB-6C4 subclones. Peripherin was also variably expressed in peripheral neuroepithelioma (NE) cell lines tested, but was organized into a more basket-like filamentous pattern in these cells. The heterogeneous expression and distribution of peripherin in NB and NE cell lines indicate that this protein is associated with maturation of the neuronal phenotype and hence serves as a differentiation marker for tumors derived from the neural crest.

Tumorigenicity and lysis by natural killers.

Detailed analysis of the natural killer (NK) activity directed at nontumorigenic cell lines and their transformed tumorigenic derivatives has revealed a paradox. On the one hand, a correlation has been found between the tumorigenic potential of chemically transformed fibroblast cell lines and their sensitivity to NK cells in vitro. Nontransformed cells (N-type cell lines) and cells tumorigenic in normal mice (C-type cell lines) are resistant to NK-mediated lysis. In contrast, cell lines that are tumorigenic in ATxFL mice (these mice are very low in NK activity), but not in normal mice (I-type cell lines) are sensitive to NK-mediated lysis. These findings support the concept that NK activity is involved in host surveillance against tumors. On the other hand, NK-resistant fibroblasts, whether taken directly form animals or derived as tumorigenic or nontumorigenic cell lines, compete with NK-sensitive target cells to inhibit their lysis by NK effectors. Not only are both NK-sensitive and -resistant cells recognized by NK effectors but both receive lytic signals from NK effector cells. Target cell resistance is a result of a protein synthesis-dependent mechanism that prevents lysis such that in the presence of inhibitors of protein synthesis all fibroblasts tested are NK sensitive. Those fibroblasts that are normally sensitive to NK-mediated lysis must be deficient in their ability to produce or respond to this counterlytic mechanism. These findings are in contrast with the general findings when lymphoid cells are studied as NK targets where sensitivity appears to be a result of recognition by NK effectors. Because our findings show that transformed and normal cells express the same recognition determinants, in order for NK activity to play an important in vivo role in tumor surveillance, a mechanism must operate to permit NK effectors to find their targets in vivo. In the absence of a special discrimination mechanism, the killing of NK-sensitive transformants that arise autochronously would be less than optimal as a consequence of competition by the normal, NK-resistant, cells.

Secretory cycle of the pituitary basophils and its morphological evidence.

The pituitary basophils of rats were examined electronmicroscopically at various time courses of the following experiments: Acute, subacute and chronic administration or perfusion of TRH and LRH separately or in combination to normal male and ovariectomized female rats as well as thyroidectomy on male rats. The findings showed a sequential, ultrastructural transformation (type I to V) of the basophil which might perform its secretory cycle. The I-type cells represent immature basophils. The II-type cells (correspond to classical TSH-cells) are angular cells in a synthesizing and/or storing phase showing an abundance of secretory granules 100-150nm in diameter. The II/III-type cells come into two categories of cell type, presenting a peripheral vesiculation of the cytoplasm. The III-type cells (correspond to classical LH-cells) are round in shape and possess a number of vesicular structures and secretory granules of about 200nm in diameter throughout the cell body. The III/IV-type cells are characterized by numerous round large cisternae which are filled with fine granulated particles. Secretory granules in these cells are 200-250nm in diameter. The enlarged spherical IV-type cells (correspond to classical FSH-cells) are packed with irregularly expanded endoplasmic reticula. Secretory granules are about 250nm in size. The IV-type cells are presumably at the end stage of granular storage or at the initiation of granular secretion, since there is a transitional cell type (IV/II-type) in which secretory granules are reduced in size (100-150 nm in diameter) and in electron density. Vesicular endoplasmic reticula are infrequently found in the IV/II-type cells, which may be either at the end of the secreting phase or at the subsequent resting phase. The V-type cells appear even in the normal and numerously after repeated or prolonged administrations of TRH or LRH. They may be exhausted basophils. It was not, however, concluded that II-, III-and IVtype cells might correspond to TSH-, LH-and FSH-cells, respectively.Based on a sequential appearance of the cells mentioned above, we propose a hypothesis: The II-type may transform into the III-type, and subsequently into the IV-type which may come back to the II-type.

Thalamic Relay Nucleus Stimulation as a Preventive Method against Recurrent Tendency following Thalamotomy for Intractable Pain

Recurrence of intractable pain was observed following basomedial thalamotomy, in spite of making a lesion as wide as possible at both the neo and paleo-spinothalamic pathway. As a last resort for prevention of recurrence, the relay nucleus stimulation for selection of the candidate among the cases suffered from intractable pain was applied in clinical cases. The mechanism of inhibitory modulating effect of relay nucleus stimulation upon the medial thalamic neuron responding to noxious stimuli was also studied in experimental animals. Experimental Results: The neurons responded to noxious stimuli in the medial thalamic nucleus are divided into 2 types; one group (F-type neuron) shows activation of action potentials with about 20 msec. latency; the other (I-type neuron) shows inhibition upon the spontaneous activities. The F-type cell is pre or post-synaptically inhibited while the I-type cell disinhibited by conditioning stimulation in the thalamic relay nucleus, although the relay nucleus does not directly connect with the medial nucleus. The inhibitory modulating effect by stimulation of the thalamic relay nucleus is suppressed following the sensori-motor cortical ablation, or making lesion in the anteromedial thalamic area. It may be concluded that the conditioning stimulation of the thalamic relay nucleus produces the inhibitory modulating effect through the cortical or intrathalamic fiber connections upon pain perceptive activity at the medial thalamic nucleus. Clinical Observations: In clinical cases whose intractable pains were suppressed by conditioning stimulation of thalamic relay nucleus, the medial thalamotomy had excellent long lasting pain killing effects. It is concluded that the pain relief effects of medial thalamotomy can be predicted before the destruction of thalamic nucleus by application of conditioning stimulation in the thalamic relay nucleus.

Identification of possible inhibitory neurons in the pericruciate cortex of the cat

The extracellular spike discharges recorded from pairs of neighboring cortical pyramidal tract and non-pyramidal tract neurons were examined for evidence which suggested inhibitory synaptic connections. The results are summarized as follows. (1) The firing frequency of some pyramidal and non-pyramidal tract neurons could be decreased by localized intracortical microiontophoretic (glutamate) stimulation of neighboring non-pyramidal tract neurons. The latter neurons, called I-type cells, could not be excited by acetylcholine. (2) In addition to the reciprocal activity patterns observed with intracortical microiontophoretic stimulation, on-line computation of joint scatter diagrams facilitated the search for 10 pairs of cells which demonstrated unidirectional negative cross-correlation functions, and included one instance where the activity of two I-type neurons was negatively correlated with that of the same pyramidal tract neuron. Latency measurements of the trough in the negative cross-correlograms revealed an onset as brief as 1.9 msec (2 pairs of cells) and as long as 7.3 msec (2 pairs of cells), with trough durations that ranged from 36.1 to 60.4 msec. Stimulation of recurrent or afferent inhibitory pathways evoked either excitation or inhibition from I-ype neurons. (3) These findings suggest that some I-type neurons may represent cortical inhibitory neurons.