Incorporating blood-based liquid biopsy information into cancer staging: time for a TNMB system?

Tissue biopsy is the standard diagnostic procedure for cancer. Biopsy may also provide material for genotyping, which can assist in the diagnosis and selection of targeted therapies but may fall short in cases of inadequate sampling, particularly from highly heterogeneous tumors. Traditional tissue biopsy suffers greater limitations in its prognostic capability over the course of disease, most obviously as an invasive procedure with potential complications, but also with respect to probable tumor clonal evolution and metastasis over time from initial biopsy evaluation. Recent work highlights circulating tumor DNA (ctDNA) present in the blood as a supplemental, or perhaps an alternative, source of DNA to identify the clinically relevant cancer mutational landscape. Indeed, this noninvasive approach may facilitate repeated monitoring of disease progression and treatment response, serving as a means to guide targeted therapies based on detected actionable mutations in patients with advanced or metastatic solid tumors. Notably, ctDNA is heralding a revolution in the range of genomic profiling and molecular mechanisms to be utilized in the battle against cancer. This review will discuss the biology of ctDNA, current methods of detection and potential applications of this information in tumor diagnosis, treatment, and disease prognosis. Conventional classification of tumors to describe cancer stage follow the TNM notation system, heavily weighting local tumor extent (T), lymph node invasion (N), and detectable metastasis (M). With recent advancements in genomics and bioinformatics, it is conceivable that routine analysis of ctDNA from liquid biopsy (B) may make cancer diagnosis, treatment, and prognosis more accurate for individual patients. We put forward the futuristic concept of TNMB tumor classification, opening a new horizon for precision medicine with the hope of creating better outcomes for cancer patients.

Concordance among gene expression-based predictors for ER-positive breast cancer treated with adjuvant tamoxifen.

BACKGROUND: ER-positive (ER+) breast cancer includes all of the intrinsic molecular subtypes, although the luminal A and B subtypes predominate. In this study, we evaluated the ability of six clinically relevant genomic signatures to predict relapse in patients with ER+ tumors treated with adjuvant tamoxifen only. METHODS: Four microarray datasets were combined and research-based versions of PAM50 intrinsic subtyping and risk of relapse (PAM50-ROR) score, 21-gene recurrence score (OncotypeDX), Mammaprint, Rotterdam 76 gene, index of sensitivity to endocrine therapy (SET) and an estrogen-induced gene set were evaluated. Distant relapse-free survival (DRFS) was estimated by Kaplan-Meier and log-rank tests, and multivariable analyses were done using Cox regression analysis. Harrell's C-index was also used to estimate performance. RESULTS: All signatures were prognostic in patients with ER+ node-negative tumors, whereas most were prognostic in ER+ node-positive disease. Among the signatures evaluated, PAM50-ROR, OncotypeDX, Mammaprint and SET were consistently found to be independent predictors of relapse. A combination of all signatures significantly increased the performance prediction. Importantly, low-risk tumors (>90% DRFS at 8.5 years) were identified by the majority of signatures only within node-negative disease, and these tumors were mostly luminal A (78%-100%). CONCLUSIONS: Most established genomic signatures were successful in outcome predictions in ER+ breast cancer and provided statistically independent information. From a clinical perspective, multiple signatures combined together most accurately predicted outcome, but a common finding was that each signature identified a subset of luminal A patients with node-negative disease who might be considered suitable candidates for adjuvant endocrine therapy alone.

Chromosomes from testicular preparations of lepidoptera.

Typical elongate, beaded chromosomes have been observed in squash preparations of testicular tissue of the butterfly Speyeria aphrodite (Fabricius), the first demonstration of relatively uncondensed chromosomes in the Lepidoptera.

Oxygen affinity in red cells: changes induced in vivo by propranolol.

Propranolol, a blocking agent for the beta adrenergic receptor, produces a redistribution of 2,3-diphosphoglycerate in the red cell. At concentrations of 3.3 x10(-5)M, 2,3-diphosphoglycerate in the red cell membrane becomes unbound in vitro. The administration of propranolol to hunmns produces similar changes and results in a decrease in the affinity of hemoglobin for oxygen.

RACK1 inhibits colonic cell growth by regulating Src activity at cell cycle checkpoints.

Previously, we showed that Src tyrosine kinases are activated early in the development of human colon cancer and are suppressed as intestinal cells differentiate. We identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src. Here we show (by overexpressing RACK1, depleting Src or RACK1 and utilizing cell-permeable peptides that perturb RACK1's interaction with Src) that RACK1 regulates growth of colon cells by suppressing Src activity at G(1) and mitotic checkpoints, and consequently delaying cell cycle progression. Activated Src rescues RACK1-inhibited growth of HT-29 cells. Conversely, inhibiting Src abolishes growth promoted by RACK1 depletion in normal cells. Two potential mechanisms whereby RACK1 regulates mitotic exit are identified: suppression of Src-mediated Sam68 phosphorylation and maintenance of the cyclin-dependent kinase (CDK) 1-cyclin B complex in an active state. Our results reveal novel mechanisms of cell cycle control in G(1) and mitosis of colon cells. The significance of this work lies in the discovery of a mechanism by which the growth of colon cancer cells can be slowed, by RACK1 suppression of an oncogenic kinase at critical cell cycle checkpoints. Small molecules that mimic RACK1 function may provide a powerful new approach to the treatment of colon cancer.

HOXA1-stimulated oncogenicity is mediated by selective upregulation of components of the p44/42 MAP kinase pathway in human mammary carcinoma cells.

Expression of homeobox A1 (HOXA1) results in oncogenic transformation of immortalized human mammary epithelial cells with aggressive tumor formation in vivo. However, the mechanisms by which HOXA1 mediates oncogenic transformation is not well defined. To identify molecules that could potentially be involved in HOXA1-mediated oncogenic transformation, microarray analysis was utilized to characterize and compare the gene expression pattern in response to forced expression or depletion of HOXA1 in human mammary carcinoma cells. Gene expression profiling identified that genes involved in the p44/42 mitogen-activated protein (MAP) kinase activation pathway (GRB2, MAP kinase kinase (MEK1) and SDFR1) or p44/42 MAP kinase-regulated genes (IER3, EPAS1, PCNA and catalase) are downstream expression targets of HOXA1. Forced expression of HOXA1 increased GRB2 and MEK1 mRNA and protein expression and increased p44/42 MAP kinase phosphorylation, activity and Elk-1-mediated transcription. Use of a MEK1 inhibitor demonstrated that increased p44/42 MAP kinase activity is required for the HOXA1-mediated increase in cell proliferation, survival, oncogenicity and oncogenic transformation. Thus, modulation of the p44/42 MAP kinase pathway is one mechanism by which HOXA1 mediates oncogenic transformation of the human mammary epithelial cell.

Micromolar Ca2+ transients in dendritic spines of hippocampal pyramidal neurons in brain slice.

The magnitude and dynamics of [Ca2+] changes in spines and dendrites of hippocampal CA1 pyramidal neurons have been characterized using a low affinity fluorescent indicator, mag-Fura 5, that is sensitive to Ca2+ in the micromolar range. During tetanic stimulation (1 s), we observed progressive [Ca2+] increases in distal CA1 spines to as much as 20-40 microM, both in organotypic slice culture and acute slice. Similar accumulations were reached during continuous depolarization (+10 mV, 1 s) when K+ channels had been blocked, but not with spike trains driven by postsynaptic current injection. The large [Ca2+] increases due to tetanic stimulation were blocked by APV, indicating that NMDA receptor-dependent influx was critical for the large responses. These findings have significant implications for low affinity Ca(2+)-dependent biochemical processes and show a new upper limit for [Ca2+] changes measured in these neurons during stimulation.

Silencing of Wnt signaling and activation of multiple metabolic pathways in response to thyroid hormone-stimulated cell proliferation.

To investigate the transcriptional program underlying thyroid hormone (T3)-induced cell proliferation, cDNA microarrays were used to survey the temporal expression profiles of 4,400 genes. Of 358 responsive genes identified, 88% had not previously been reported to be transcriptionally or functionally modulated by T3. Partitioning the genes into functional classes revealed the activation of multiple pathways, including glucose metabolism, biosynthesis, transcriptional regulation, protein degradation, and detoxification in T3-induced cell proliferation. Clustering the genes by temporal expression patterns provided further insight into the dynamics of T3 response pathways. Of particular significance was the finding that T3 rapidly repressed the expression of key regulators of the Wnt signaling pathway and suppressed the transcriptional downstream elements of the beta-catenin-T-cell factor complex. This was confirmed biochemically, as beta-catenin protein levels also decreased, leading to a decrease in the transcriptional activity of a beta-catenin-responsive promoter. These results indicate that T3-induced cell proliferation is accompanied by a complex coordinated transcriptional reprogramming of many genes in different pathways and that early silencing of the Wnt pathway may be critical to this event.

High-fidelity mRNA amplification for gene profiling.

The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.

Iron addiction: a novel therapeutic target in ovarian cancer.

Ovarian cancer is a lethal malignancy that has not seen a major therapeutic advance in over 30 years. We demonstrate that ovarian cancer exhibits a targetable alteration in iron metabolism. Ferroportin (FPN), the iron efflux pump, is decreased, and transferrin receptor (TFR1), the iron importer, is increased in tumor tissue from patients with high grade but not low grade serous ovarian cancer. A similar profile of decreased FPN and increased TFR1 is observed in a genetic model of ovarian cancer tumor-initiating cells (TICs). The net result of these changes is an accumulation of excess intracellular iron and an augmented dependence on iron for proliferation. A forced reduction in intracellular iron reduces the proliferation of ovarian cancer TICs in vitro, and inhibits both tumor growth and intraperitoneal dissemination of tumor cells in vivo. Mechanistic studies demonstrate that iron increases metastatic spread by facilitating invasion through expression of matrix metalloproteases and synthesis of interleukin 6 (IL-6). We show that the iron dependence of ovarian cancer TICs renders them exquisitely sensitive in vivo to agents that induce iron-dependent cell death (ferroptosis) as well as iron chelators, and thus creates a metabolic vulnerability that can be exploited therapeutically.

BDNF enhances quantal neurotransmitter release and increases the number of docked vesicles at the active zones of hippocampal excitatory synapses.

Brain-derived neurotrophic factor (BDNF) is emerging as a key mediator of activity-dependent modifications of synaptic strength in the CNS. We investigated the hypothesis that BDNF enhances quantal neurotransmitter release by modulating the distribution of synaptic vesicles within presynaptic terminals using organotypic slice cultures of postnatal rat hippocampus. BDNF specifically increased the number of docked vesicles at the active zone of excitatory synapses on CA1 dendritic spines, with only a small increase in active zone size. In agreement with the hypothesis that an increased docked vesicle density enhances quantal neurotransmitter release, BDNF increased the frequency, but not the amplitude, of AMPA receptor-mediated miniature EPSCs (mEPSCs) recorded from CA1 pyramidal neurons in hippocampal slices. Synapse number, independently estimated from dendritic spine density and electron microscopy measurements, was also increased after BDNF treatment, indicating that the actions of BNDF on mEPSC frequency can be partially attributed to an increased synaptic density. Our results further suggest that all these actions were mediated via tyrosine kinase B (TrkB) receptor activation, established by inhibition of plasma membrane tyrosine kinases with K-252a. These results provide additional evidence of a fundamental role of the BDNF-TrkB signaling cascade in synaptic transmission, as well as in cellular models of hippocampus-dependent learning and memory.

Impairments in high-frequency transmission, synaptic vesicle docking, and synaptic protein distribution in the hippocampus of BDNF knockout mice.

Brain-derived neurotrophic factor (BDNF) promotes long-term potentiation (LTP) at hippocampal CA1 synapses by a presynaptic enhancement of synaptic transmission during high-frequency stimulation (HFS). Here we have investigated the mechanisms of BDNF action using two lines of BDNF knockout mice. Among other presynaptic impairments, the mutant mice exhibited more pronounced synaptic fatigue at CA1 synapses during high-frequency stimulation, compared with wild-type animals. Quantitative analysis of CA1 synapses revealed a significant reduction in the number of vesicles docked at presynaptic active zones in the mutant mice. Synaptosomes prepared from the mutant hippocampus exhibited a marked decrease in the levels of synaptophysin as well as synaptobrevin [vesicle-associated membrane protein (VAMP-2)], a protein known to be involved in vesicle docking and fusion. Treatment of the mutant slices with BDNF reversed the electrophysiological and biochemical deficits in the hippocampal synapses. Taken together, these results suggest a novel role for BDNF in the mobilization and/or docking of synaptic vesicles to presynaptic active zones.

A phase II clinical trial of weekly paclitaxel and carboplatin in combination with panitumumab in metastatic triple negative breast cancer.

PURPOSE: Women with metastatic triple negative breast cancer (TNBC) can have a poor prognosis with treatment limited to cytotoxic chemotherapy. The identification of effective therapies that may limit exposure to cytotoxic chemotherapy and lead to prolonged survival is an unmet medical need. We tested an inhibitor of the epidermal growth factor receptor, panitumumab in combination with chemotherapy. METHODS: We conducted a single arm clinical trial in women with metastatic or locally advanced TNBC to paclitaxel 80 mg/m2 and carboplatin AUC of 2 on days 1, 8, and 15 and panitumumab 6 mg/kg on days 1 and 15 for a cycle length of 28 days. The objectives were to evaluate the response rate and safety of the combination in comparison to historical controls. RESULTS: Fourteen patients with TNBC were enrolled with a median age of 53 years. The majority of women were African American (64.3%) with visceral metastasis (64.2%). Hematologic toxicities, particularly neutropenia and thrombocytopenia, were a major cause of missed chemotherapy and delayed treatment in this study. The overall response rate (complete and partial response) of the 13 evaluable patients was 46%. The median time to best response was 2.4 months and the median time to disease progression was 3.6 months. We were able to perform the PAM50 analysis on tumors from 7 of our subjects. All the samples tested clustered within the basal-like subtype. CONCLUSIONS: In our experience the response rate of carboplatin, paclitaxel and panitumumab was consistent with other reports of response for cytotoxic chemotherapy in metastatic TNBC.

Spontaneous pyramidal cell death in organotypic slice cultures from rat hippocampus is prevented by glutamate receptor antagonists.

A predictable pattern of selective neuronal cell death occurs in organotypic slice cultures of neonatal rat hippocampus during the second and third weeks in vitro. We serially examined organotypic cultures at three, four, seven, 14, 21 and 28 days in vitro, using uptake of the fluorescent dye propidium iodide to identify degenerating cells. After seven days in vitro, the cell degeneration that accompanies the slicing procedure appears to have ended. However, at 14 days in vitro, degenerating neurons could be identified in area CA3. When many degenerating cells were present in a slice, they were distributed in the dentate hilus (CA4) and proximal portions of CA1 as well. Neuronal degeneration involving mainly CA1 pyramidal cells was still apparent at 21 days in vitro, but was much less marked than at 14 days. Study of fixed cultures with light and electron microscopy methods confirmed the presence of degenerating neurons with a pyknotic or vacuolated appearance. Spontaneous neuronal degeneration at 14 and at 21 days in vitro was almost entirely prevented by the addition of 10.5 mM Mg2+ or 3 mM kynurenic acid (a glutamate receptor antagonist), beginning at seven days in vitro. Cell death was markedly decreased by treatment with 100 microM DL-2-amino-5-phosphonovaleric acid (a selective antagonist of N-methyl-D-aspartate glutamate receptors). Removal of the blocking agents by returning cultures to control media at 28 days in vitro induced widespread neuronal degeneration, involving all the regions of the hippocampal slice cultures. The inhibition of spontaneous neuronal cell death by glutamate receptor antagonists and by blockade of glutamate release at synapses suggests that the mechanism of cell death involves glutamate receptors. The time course of degeneration suggests that the vulnerability to glutamate excitotoxicity is an aspect of developmentally regulated components of glutamatergic synapses acquired in the hippocampal organotypic cultures after the first week in vitro.

Calcium/calmodulin-dependent protein kinase II clusters in adult rat hippocampal slices.

We have previously reported the formation of calcium/calmodulin-dependent protein kinase II (CaMKII) clusters approximately 110 nm in diameter in hippocampal neurons in culture and in the intact adult brain, under conditions that simulate ischemic stress and increase [Ca(2+)](i) [Dosemeci et al. (2000) J. Neurosci. 20, 3076-3084; Tao-Cheng et al. (2001) Neuroscience 106, 69-78]. These observations suggest that ischemia-like conditions that prevail during the dissection of brain tissue for the preparation of hippocampal slices could lead to the formation of CaMKII clusters. We now show by pre-embedding immuno-electron microscopy that, indeed, CaMKII clusters are present in the CA1 pyramidal neurons in hippocampal slices from adult rats fixed immediately after dissection, and that the number of CaMKII clusters increases with the delay time between decapitation and fixation. Moreover, CaMKII clusters are typically localized near the endoplasmic reticulum. When acute slices are allowed to recover in oxygenated medium for 2 h, CaMKII clusters mostly disappear, indicating that clustering is reversible. Also, the postsynaptic density, another site for CaMKII accumulation under excitatory conditions, becomes thinner upon recovery. Treatment of recovered slices with high potassium for 90 s causes the re-appearance of CaMKII clusters in nearly all CA1 pyramidal cells examined. On the other hand, when dissociated hippocampal neurons in primary culture are exposed to the same depolarizing conditions, only approximately 25% of neurons exhibit CaMKII clusters, indicating a difference in the susceptibility of the neurons in culture and in acute slices to excitatory stimuli. Altogether these observations indicate that the effect of CaMKII clustering should be considered when interpreting experimental results obtained with hippocampal slices.

Spontaneous and evoked glutamate signalling influences Fos-lacZ expression and pyramidal cell death in hippocampal slice cultures from transgenic rats.

Previously, we established that a spatially and temporally predictable pattern of spontaneous cell death occurs in pyramidal hippocampal neurons maintained in organotypic slice cultures. We have begun to examine the signalling events that may be relevant to this process by analyzing the expression of cellular immediate-early genes (cIEGs). In the present studies, organotypic hippocampal cultures were generated from transgenic rats that carry a fos-lacZ fusion gene. beta-Galactosidase activity in these rats accurately recapitulates Fos expression. An association was observed between cell death, as determined by propidium iodide (PI) staining, and Fos-lacZ expression. There was a consistent rise in beta-galactosidase activity in vulnerable regions 1-2 days before the peak of spontaneous neuronal death. Long-term treatment with TTX, CNQX, or D,L-APV inhibited the spontaneous neuronal death as well as Fos-lacZ expression. Furthermore, Fos-lacZ induction and cell death could be evoked by removal of these receptor antagonists or by application of the excitotoxin, kainic acid. The association between cIEG expression and cell death, shown here and by others, suggests that these genes contribute to regulatory events involved with cell death and/or protection.

Correlated measurements of free and total intracellular calcium concentration in central nervous system neurons.

Transient changes in the intracellular concentration of free calcium ([Ca2+])i) act as a trigger or modulator for a large number of important neuronal processes. Such transients can originate from voltage- or ligand-gated fluxes of Ca2+ into the cytoplasm from the extracellular space, or by ligand- or Ca2+(-)gated release from intracellular stores. Characterizing the sources and spatio-temporal patterns of [Ca2+]i transients is critical for understanding the role of different neuronal compartments in dendritic integration and synaptic plasticity. Optical imaging of fluorescent indicators sensitive to free Ca2+ is especially suited to studying such phenomena because this approach offers simultaneous monitoring of large regions of the dendritic tree in individual living central nervous system neurons. In contrast, energy-dispersive X-ray (EDX) microanalysis provides quantitative information on the amount and location of intracellular total, i.e., free plus bound, calcium (Ca) within specific subcellular dendritic compartments as a function of the activity state of the neuron. When optical measurements of [Ca2+]i transients and parallel EDX measurements of Ca content are used in tandem, and correlated simultaneously with electrophysiological measurements of neuronal activity, the combined information provides a relatively general picture of spatio-temporal neuronal total Ca fluctuations. To illustrate the kinds of information available with this approach, we review here results from our ongoing work aimed at evaluating the role of various Ca uptake, release, sequestration, and extrusion mechanisms in the generation and termination of [Ca2+]i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. Our observations support the long-standing speculation that the dendritic endoplasmic reticulum acts not only as an intracellular Ca2+ source that can be mobilized by a signal cascade originating at activated synapses, but also as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca2+ influx.

Improved renal function and inhibition of renin and aldosterone secretion following peritoneovenous (LeVeen) shunt.

Twelve patients with cirrhosis, refractory ascites, and varying degrees of renal failure (creatinine clearance, 5 to 44 ml/min) were studied before and up to 2 weeks following peritoneovenous shunt. Creatinine clearance increased 60% or more in seven patients (group I) and 22% or less in five patients (group II). There were no significant differences in maximum urine output or sodium excretion between groups (group I, 4,272 ml/14 hr, 372 mEq/24 hr; group II, 3,722 ml/24 hr, 255 mEq/24 hr). Aldosterone and renin concentrations were higher in group I and showed a greater decrease after shunting. Renin substrate levels also were higher in group I and rose following shunt insertion, while group II remained low. Ascitic fluid was found to contain renin substrate in concentrations of approximately 25% to 50% of plasma concentrations. Patients with the greatest increase in creatinine clearance showed the largest rise in substrate concentration and fall in renin and aldosterone secretion, suggesting a dynamic relationship between these factors. That a diuresis could occur without significant change in these parameters in five of 12 patients suggests independent control mechanisms for renal salt and water excretion and glomerular filtration in the ascitic patient.

Prevention of increased hemoglobin-oxygen affinity in open-heart operations with inosine-phosphate-pyruvate solution.

In a control group of 32 patients undergoing open-heart operation, erythrocyte 2,3-diphosphoglycerate (2,3-DPG) declined progressively during the course of perfusion from a prebypass mean of 17.00 to 11.29 mu M per gram of hemoglobin at the end of bypass. The decrease was greater than that attributable merely to dilution of the patients' cells with the 2,3-DPG-deficient donor cells used to prime the pump oxygenator circuit. Administration of 300 mg of allopurinol, to prevent the conversion of inosine to uric acid, every 8 hours during the 24 hours prior to operation in 11 patients did not prevent the 2,3-DPG decrease during heart-lung bypass: prebypass, 18.31; postbypass, 13.56 mu M/gm Hgb. The mean P50 for both these groups combined decreased from a prebypass mean of 25.7 to a postbypass level of 21.9 torr. A solution of 0.1 M inosine, 0.1 M pyruvate, and 0.066 M phosphate (IPP) in a dosage of 7.5 ml per kologram of body weight prevented the 2,3-DPG decrease: prebypass, 15.74; postbypass, 14.85. Administration of 15 ml per kilogram of IPP in 15 patients preserved 2,3-DPG: prebypass, 18.09; postbypass, 18.52. The P50 remained unchanged in this last group. The method of providing for myocardial oxygen requirements during bypass was not standardized, and therefore the protective effect of IPP against ischemic damage in patients undergoing aortic valve replacement or myocardial revascularization could not be evaluated. No deleterious effects of IPP were noted.

Long-term potentiation-induced synaptic changes in hippocampal dentate gyrus of rats with an inborn low or high learning capacity.

Structural changes induced by high frequency (HF) stimulation on granule cells of the hippocampal dentate gyrus of rats with inborn high (HP) and low (LP) learning capacity was examined in this study. Two types of axodendritic synapses were processed morphometrically; the axonal bouton on dendritic shafts and those making contact through dendritic spines. The occurrence of granule cell spine synapses on LP rats before tetanus is less than the one in control animals. However, after long-term potentiation (LTP) produced by HF stimulation the number of spine synapses decreased, as happened in the control rats. Moreover, the number of granule cells shaft synapses counted on LP rats, before tetanus is less than the one on control rats, and after LTP this number increased greatly. There was no change in the number of granule cell shaft synapses in HP rats before and after LTP. LP rats which did not show LTP in spite of HF stimulation did not show changes in the number of spine and shaft synapses. From these results we can conclude that: (1) the great hippocampal synaptic plasticity observed in HP rats compared to LP rats can be attributed, at least in part, to the increased number of spine and shaft synapses observed in HP rats; (2) the minor number of spine and shaft synapses in LP rats may account for the impossibility to obtain LTP after HF stimulation in these animals.