Tau and amyloid-related pathologies in the entorhinal cortex have divergent effects in the hippocampal circuit.

The entorhinal cortex (EC) is affected early in Alzheimer's disease, an illness defined by a co-occurrence of tau and amyloid-related pathologies. How the co-occurrence of these pathologies in the EC affects the hippocampal circuit remains unknown. Here we address this question by performing electrophysiological analyses of the EC circuit in mice that express mutant human amyloid precursor protein (hAPP) or tau (hTau), or both in the EC. We show that the alterations in the hippocampal circuit are divergent, with hAPP increasing but hTau decreasing neuronal/circuit excitability. Most importantly, mice co-expressing hAPP and hTau show that hTau has a dominant effect, dampening the excitatory effects of hAPP. Additionally, compensatory synaptic downscaling, in response to increased excitability in EC was observed in subicular neurons of hAPP mice. Based on simulations, we propose that EC interneuron pruning can account for both EC hyperexcitability and subicular synaptic downscaling found in mice expressing hAPP.

Lymphocyte infiltration of neocortex and hippocampus after a single brief seizure in mice.

Various immune responses have been described in epileptic patients and animal models of epilepsy, but immune responses in brain after a single seizure are poorly understood. We studied immune responses in brain after a single brief generalized tonic-clonic seizure in mice. C57bl/6 mice, either unanesthetized or anesthetized (pentobarbital, ethyl chloride) received either electrical (15-30 mA, 100 Hz, 1s) or sham stimulation (subcutaneous electrodes over frontal lobe, no current). Electrical stimulation of unanesthetized mice resulted in tonic-clonic convulsions with hind-limb extension (maximal seizure), tonic-clonic convulsions without hind-limb extension (submaximal seizure), or no seizure. In contrast, such stimulation of anesthetized mice did not result in seizure. Mice were killed at 1h-7 days after seizure. Brains or regions dissected from brain (neocortex, hippocampus, midbrain, cerebellum) of each group were pooled, single cell suspensions prepared, and cells separated according to density. CD4(+) (CD3(+)CD45(Hi)) and CD8(+) (CD3(+)CD45(Hi)) T cell and CD45R(+) (CD45(Hi)) B cell numbers were determined by flow cytometry. At 24h after a maximal seizure, CD4(+) and CD8(+) T cells and CD45R(+) B cells appeared in brain, reaching peak numbers at 48 h, but were no longer detected at 7days. CD4(+) T cells and CD45R(+) B cells were preferentially found in neocortex compared with hippocampus, whereas CD8(+) T cells were preferentially found in hippocampus at 24h after a maximal seizure. In contrast, virtually no lymphocytes were detected in brains of unstimulated or sham stimulated mice, unanesthetized stimulated mice after submaximal or no seizure, and anesthetized stimulated mice at 1 h-7 day. Neither Ly6-G+ neutrophils nor erythrocytes were detected in brains of any animals, nor was there any detectable increase of blood-brain barrier permeability by uptake of Evans Blue dye. The results indicate that lymphocyte entry into brain after a single brief seizure is due to a selective process of recruitment into cortical regions.

Local axon collaterals of area CA1 support spread of epileptiform discharges within CA1, but propagation is unidirectional.

CA3 and subiculum are hippocampal formation regions that can initiate seizure activity because each has a substantial intrinsic excitatory connectivity. We studied the intrinsic connectivity of area CA1 by exploring the spread of synchronous population discharges in ventral hippocampal slices from rats using a recording chamber that permitted multiple simultaneous extracellular recordings along all laminae of CA1. Brief single stimulus pulses were applied to stratum oriens (SO) or stratum radiatum (SR) on the CA3 side or the subicular side of CA1. In disinhibited slices, events triggered with SO or SR stimulation on the CA3-side propagated over the proximo-distal extent of CA1 with a maximal conduction velocity of 0.4 m/s, comparable with antidromic conduction velocities within CA1. By contrast, SO or SR stimuli applied on the subicular side of CA1 triggered events that did not spread "backward" toward CA3. These events are rapidly decremented in amplitude and duration. Whereas antidromic responses were largest when stimuli were applied on the subicular side of CA1, such responses were not sufficient to trigger epileptiform discharges when excitatory transmission was intact. We conclude that the unidirectional spread of epileptiform activity in area CA1 is the result of an intrinsic axon collateral system where each pyramidal cell has a proportionally larger projection toward subiculum. Although this collateral system is sparse compared with other hippocampal formation regions, its unidirectionality protects against re-entrant activation of CA3 and may be physiologically significant as a relay from proximal CA1 to distal CA1.

Hemispheric differences in protein kinase C betaII levels in the rat amygdala: baseline asymmetry and lateralized changes associated with cue and context in a classical fear conditioning paradigm.

The amygdala is critically important for fear learning, and specific kinases have been implicated as contributors to the mechanisms that underlie learning. We examined levels of protein kinase C betaII (PKC betaII) in the left and right lateral and basolateral nuclei (LA/BLA) of the amygdala from animals that were classically fear conditioned with tones as cues and footshocks. Groups consisted of animals that received neither tones nor shocks, paired tones and shocks, or unpaired tones and shocks. At 1 h after conditioning, some animals from each group were used for biochemical measurements of PKC betaII levels and other animals were given probe trials to assess freezing behavior to cue and context. The levels of PKC betaII were greater in the left hemisphere in animals receiving neither tones nor shocks and animals receiving paired tones and shocks. PKC betaII levels were greater in the right hemisphere of animals receiving randomly presented tones and shocks. Freezing times to cue were long (>80% of probe trial time) in both the paired tone/shock and randomly unpaired tone/shock groups. Freezing times to context were long in the unpaired tone/shock group, but not the paired tone/shock group. Correlational analyses showed that freezing times to context, but not cue, precisely predicted the right/left relation of PKC betaII levels in the LA/BLA: the greater the time spent freezing to context, the greater the increase in right hemisphere PKC betaII levels. We conclude that fear conditioning causes hemisphere and input specific increases in PKC betaII in the rat LA/BLA.

Methods and insights from the characterization of osteoprogenitor cells of bats (Mammalia: Chiroptera).

Osteoprogenitor cells contribute to the development and maintenance of skeletal tissues. Bats are unique model taxa whose cellular processes are poorly understood, especially in regards to skeletal biology. Forelimb bones of bats, unlike those of terrestrial mammals, bend during flight and function in controlled deformation. As a first step towards understanding the molecular processes governing deposition of this flexible bone matrix, we provide the first method for isolation and differentiation of cell populations derived from the bone marrow and cortical bone of bats, and compare results with those harvested from C57BL/6J mice. Osteogenic capacity of these cells was assessed via absolute quantitative real-time PCR (qPCR) and through quantification of in vitro mineral deposition. Results indicate the differentiated bone cells of bats display significantly lower gene expression of known osteogenic markers (Runt-related transcription factor (RUNX2), osteocalcin (BGLAP) and osterix (SP7)), and deposit a less-mineralized matrix compared with murine controls. By characterizing the in vitro performance of osteoprogenitor cells throughout differentiation and matrix production, this study lays the ground work for in vitro manipulations of bat stem and osteoprogenitor cells and extends our understanding of the cellular diversity across mammals that occupy different habitats.

Laryngospasm, central and obstructive apnea during seizures: Defining pathophysiology for sudden death in a rat model.

Seizure spread into the autonomic nervous system can result in life-threatening cardiovascular and respiratory dysfunction. Here we report on a less-studied consequence of such autonomic derangements-the possibility of laryngospasm and upper-airway occlusion. We used parenteral kainic acid to induce recurring seizures in urethane-anesthetized Sprague Dawley rats. EEG recordings and combinations of cardiopulmonary monitoring, including video laryngoscopy, were performed during multi-unit recordings of recurrent laryngeal nerve (RLN) activity or head-out plethysmography with or without endotracheal intubation. Controlled occlusions of a tracheal tube were used to study the kinetics of cardiac and respiratory changes after sudden obstruction. Seizure activity caused significant firing increases in the RLN that were associated with abnormal, high-frequency movements of the vocal folds. Partial airway obstruction from laryngospasm was evident in plethysmograms and was prevented by intubation. Complete glottic closure (confirmed by laryngoscopy) occurred in a subset of non-intubated animals in association with the largest increases in RLN activity, and cessation of airflow was followed in all obstructed animals within tens of seconds by ST-segment elevation, bradycardia, and death. Periods of central apnea occurred in both intubated and non-intubated rats during seizures for periods up to 33s and were associated with modestly increased RLN activity, minimal cardiac derangements, and an open airway on laryngoscopy. In controlled complete airway occlusions, respiratory effort to inspire progressively increased, then ceased, usually in less than 1min. Respiratory arrest was associated with left ventricular dilatation and eventual asystole, an elevation of systemic blood pressure, and complete glottic closure. Severe laryngospasm contributed to the seizure- and hypoxemia-induced conditions that resulted in sudden death in our rat model, and we suggest that this mechanism could contribute to sudden death in epilepsy.

Flaccid quadriparesis associated with Yersinia enterocolitis-induced hypokalemia.

A 23-year-old woman presented with diarrhea, flaccid quadriparesis, a low serum potassium level, and an elevated creatine kinase level. A stool culture yielded Yersinia enterocolitica, and a muscle biopsy was compatible with a hypokalemic myopathy. The patient's diarrhea responded to sulfamethoxazole and trimethoprim therapy. We suggest that Y enterocolitica be added to the group of intestinal pathogens capable of producing hypokalemia and rhabdomyolysis.

Isolation, purification, and partial characterization of an autocrine periodontal ligament cell chemotactic factor.

Periodontal ligament (PDL) cells are believed to play a critically important role in the regeneration of the periodontium. We have suggested that polypeptide growth factors can enhance periodontal regeneration by stimulating PDL cell chemotaxis and mitogenesis. This manuscript describes the identification of a novel chemotactic factor isolated from human PDL cells which we named PDL-CTX. PDL-CTX induces the directed migration of human PDL cells in vitro and was found to be a more potent chemotactic agent than other known growth factors. Additionally, PDL-CTX has no chemotactic effect on gingival fibroblasts or gingival epithelial cells. Both tryptic digestion and boiling abolished PDL-CTX's biological activity. The designed purification method included Mono-S cation exchange, heparin-sepharose affinity, and microbore reverse-phase HPLC. The purified factor has a relative molecular weight of approximately 7000 daltons based on sodium dodecyl sulfate (SDS) gel analysis. The amino acid composition and partial amino acid sequence were determined from HPLC-purified material. These were determined to be unique. Further investigation of the biological functions of PDL-CTX on PDL cells and other ligament cells should help improve our understanding of ligament repair.

Mitogenic and chemotactic responses of human periodontal ligament cells to the different isoforms of platelet-derived growth factor.

A major focus of studies that center on regeneration of the periodontium is to determine the efficacy of the use of polypeptide growth factors. Platelet-derived growth factor has been reported to be a possible agent for clinical use. PDGF has various isoforms. Therefore, we decided to study the mitogenic and chemotactic responses of human periodontal ligament (PDL) cells to recombinant human PDGF-AB, AA, and BB. Addition of each isoform of PDGF to in vitro mitogenesis assays induced PDL cell proliferation in a dose-dependent manner. The maximum mitogenic effect was evident at the concentration of 100 ng/mL. In these assays, PDGF-BB was found to be the most potent mitogen. PDGF-AB elicited an intermediate response, and PDGF-AA was the least effective. The results of chemotaxis assays closely parallel those of the mitogenesis assays. PDGF-BB exhibited the most potent chemotactic effect. The maximal effect was observed at 10 ng/mL. The findings of these experiments indicate that PDGF-BB is more effective than the other isoforms in promoting mitogenesis and chemotaxis of PDL cells in vitro, and may therefore be a suitable ethical pharmaceutical for use in periodontal regeneration procedures.

Expression of PrP mRNA is regulated by a fragment of MRP8 in human fibroblasts.

Prion protein (PrPc) is expressed in many tissues, both in human and animals. The scrapie isoform of PrPc has been shown to cause neurodegeneration. In other studies it has been demonstrated that overexpression of the PrP gene can result in nonneuronal tissue degradation. Little is known, however, about the normal function of PrPc and prion protein gene regulation. Using cultured periodontal ligament cells as an experimental model, we have demonstrated the stimulation of PrP mRNA expression by MRP8 (migration inhibitory factor-related protein). Additionally, we have shown that PDGF has an opposite effect acting as a suppresser. We propose that a correlation exists between PrPc mRNA expression and cell growth arrest and differentiation.

Sex differences in anxiety, sensorimotor gating and expression of the alpha4 subunit of the GABAA receptor in the amygdala after progesterone withdrawal.

In a progesterone withdrawal (PWD) model of premenstrual anxiety, we have previously demonstrated that increased hippocampal expression of the alpha4 subunit of the GABAA receptor (GABAA-R) is closely associated with higher anxiety levels in the elevated plus maze. However, several studies indicate that sex differences in regulation of the GABAA-R in specific brain regions may be an important factor in the observed gender differences in mood disorders. Thus, we investigated possible sex differences in GABAA-R subunit expression and anxiety during PWD. To this end, we utilized the acoustic startle response (ASR) to assess anxiety levels in male and female rats undergoing PWD as the ASR is also applicable to the assessment of human anxiety responses. We also investigated GABAA-R alpha4 subunit expression in the amygdala, as the amygdala directly regulates the primary startle circuit. Female rats exhibited a greater ASR during PWD than controls, indicating higher levels of anxiety and arousal. In contrast, male rats undergoing PWD did not demonstrate an increased ASR. The sex differences in the ASR were paralleled by sex differences in the expression of the GABAA-R alpha4 subunit in the amygdala such that alpha4 subunit expression was up-regulated in females during PWD whereas alpha4 levels in males undergoing PWD were not altered relative to controls. These findings might have implications regarding gender differences in human mood disorders and the aetiology of premenstrual anxiety.

Comparison of in vitro proliferative capacity of human periodontal ligament cells in juvenile and aged donors.

OBJECTIVE: The aim of this study is to compare the in vitro proliferative capacity of periodontal ligament (PDL) cells from aged and juvenile donors. MATERIALS AND METHODS: Flow-cytometric analysis of the cell cycle was used to compare the length of each cell cycle, and the ratio of the cells progressing through the cycles between four PDL cells from juvenile donors and four cells from aged donors. Then, replicative capacity of the PDL cells from three juvenile and three aged donors was compared by serial cultures. Finally, expression of c-fos was compared between cells proliferating and cells which had reached senescent. RESULTS: Flow-cytometric analysis of the cell cycle had revealed that although there were no differences in the length of each phase of the cell cycle, significant differences were found in the ratio of the cells entering from Gap I to DNA synthesis phase of the cell cycle (P < 0.025). Replicative capacity was much longer in two cells from juvenile donors (about 20 population doublings), while all cells from aged donors showed short dividing abilities (less than eight population doublings), hence entered senescent phases shortly. Additionally, no c-fos was detected in cells which had reached senescence upon stimulation with serum. CONCLUSIONS: It is generally believed that aged humans have an impaired wound healing ability. We believe that more fibrotic PDL tissues seen in aged humans might be the reason for this, and suggest that this phenomena might be due to the progressive accumulation of senescent cell populations.