Canine distemper virus induces apoptosis in cervical tumor derived cell lines

Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV) induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi), by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control

Origin and Properties of Striatal Local Field Potential Responses to Cortical Stimulation: Temporal Regulation by Fast Inhibitory Connections

Evoked striatal field potentials are seldom used to study corticostriatal communication in vivo because little is known about their origin and significance. Here we show that striatal field responses evoked by stimulating the prelimbic cortex in mice are reduced by more than 90% after infusing the AMPA receptor antagonist CNQX close to the recording electrode. Moreover, the amplitude of local field responses and dPSPs recorded in striatal medium spiny neurons increase in parallel with increasing stimulating current intensity. Finally, the evoked striatal fields show several of the basic known properties of corticostriatal transmission, including paired pulse facilitation and topographical organization. As a case study, we characterized the effect of local GABAA receptor blockade on striatal field and multiunitary action potential responses to prelimbic cortex stimulation. Striatal activity was recorded through a 24 channel silicon probe at about 600 µm from a microdialysis probe. Intrastriatal administration of the GABAA receptor antagonist bicuculline increased by 65±7% the duration of the evoked field responses. Moreover, the associated action potential responses were markedly enhanced during bicuculline infusion. Bicuculline enhancement took place at all the striatal sites that showed a response to cortical stimulation before drug infusion, but sites showing no field response before bicuculline remained unresponsive during GABAA receptor blockade. Thus, the data demonstrate that fast inhibitory connections exert a marked temporal regulation of input-output transformations within spatially delimited striatal networks responding to a cortical input. Overall, we propose that evoked striatal fields may be a useful tool to study corticostriatal synaptic connectivity in relation to behavior

Evoked striatal field potentials show paired pulse facilitation.

<p><b>A.</b> Amplitude of the striatal field response (mean±SEM, n = 5) as a function of stimulation current intensity for cortical paired pulse stimulation (interstimulus interval 50 ms). <b>B.</b> Simultaneous recordings of striatal eLFP through a glass micropipette (<i>above</i>) and MSN membrane potential (<i>below</i>) after cortical paired pulse stimulation. <b>C–D.</b> Paired pulse stimulation at the prelimbic cortex (400 µA and 50 ms interstimulus interval) induces a facilitation of the response to the second stimulus in MSNs (C, *<i>p</i><0.0001, Student's paired t test, n = 7) and evoked striatal field potentials (D, *<i>p</i><0.005, Student's paired t test, n = 7).</p

Striatal field responses are related to dPSPs in medium spiny neurons.

<p><b>A.</b> As in rats, the membrane potential of mouse MSNs alternates between up and down states. The histogram (1 mV bin) shows a biphasic distribution of Vm values corresponding to 20 seconds of the illustrated intracellular recording. <b>B.</b> Simultaneous intracellular (<i>above</i>) and local field (<i>below</i>) recordings obtained with separate glass microelectrodes after stimulating the prelimbic cortex with different current intensities. Individual traces are displayed in gray, averages in black. The through in the local field response coincides temporally with the peak of the dPSP. <b>C.</b> The amplitudes of simultaneously recorded striatal dPSPs and evoked local field potentials (eLFP) increase in parallel with increasing stimulation intensities. Data corresponds to one simultaneously recorded pair stimulated at 200, 400 and 600 µA. <b>D.</b> Correlation between eLFP and dPSP amplitude evoked by prelimbic cortex stimulation at 400 µA in five different experiments (n = 5, r² = 0.94, <i>p</i><0.01).</p

Intrastriatal bicuculline increases the amplitude and duration of striatal output.

<p><b>A.</b> Representative multiunitary response to cortical stimulation before, during and after delivering bicuculline into the striatum. The top trace corresponds to the rectified, smoothed and averaged action potential activity of 20 individual trials. Dotted line reflects 3 SD of multiunitary activity during 100 ms prior to stimulation onset (400 µA). Note the increase in the amplitude and duration of the response after bicuculline. Similar results were obtained for 300 µA cortical stimulation. <b>B–D.</b> Effect of bicuculline on the area (<b>B</b>), amplitude (<b>C</b>) and duration (<b>D</b>) of multiunitary action potential responses of 20 recording sites at the hot spot from 3 different experiments. * <i>p</i><0.0001, Wilcoxon paired test. # <i>p</i><0.0001 Student's paired t test.</p

Local administration of CNQX blocks synaptic responses in the striatum.

<p><b>A.</b> Histological sections showing the location of the multichannel electrode (left) and microdialysis probe (right) in a representative experiment. <b>B.</b> Striatal field responses evoked by stimulating the prelimbic cortex remained stable for hours under continuous infusion of ACSF through the microdyalisis probe (n = 3). <b>C.. </b><i>Left:</i> Time course of the blocking effect of CNQX on striatal field responses evoked by cortical stimulation in a representative experiment. Each point is the amplitude of a single evoked response, which was evaluated every ten seconds during the experiment. The superimposed line corresponds to the smoothed data with a moving average window (20 samples). <i>Right:</i> Field potential responses corresponding to the individual trials depicted in gray, and multiunit activity recorded from the same electrode contact, before, after 10 minutes of CNQX infusion, and after 40 minutes of washing out with ACSF. Data are from the experiment shown at the left. <b>D.</b> CNQX administration (200 µM) almost completely blocked the striatal field response to cortical stimulation (* <i>p</i><0.001, Tukey post-hoc test, repeated measures ANOVA, N = 4). <b>E.. </b><i>Left:</i> Histological reconstruction showing the location of three recording sites in the multichannel electrode (<i>a, b, c</i>) and the microdialysis probe in one of the CNQX experiments. <i>Middle:</i> Time course of CNQX effect at sites <i>a, b</i> and <i>c</i>. Note that CNQX reached first the closest recording site <i>a</i> than the more distant sites <i>b</i> and <i>c</i>. Distance between illustrated recording sites: 400 µm. <i>Right:</i> Striatal field response during the first 5 minutes and at the 10th minute of CNQX infusion in the same experiment, showing that by the fourth minute, response in site <i>a</i> was abolished but remained almost unchanged 800 µm away in site <i>c</i>. Note that the time course of CNQX effect depends on the location of the recording electrode and not on the basal amplitude of the response (compare site <i>a</i> with site <i>c</i>).</p

Local administration of a GABA_A receptor antagonist increases striatal evoked field potential responses.

<p><b>A.</b> Time course of the evoked field potential amplitude of a representative striatal site tested with 300 µA stimulation intensity. Note a ∼50% amplitude increase after 10 minutes of bicuculline infusion. <b>B.</b> Time course of the evoked field potential amplitude of a representative striatal site tested with 400 µA stimulation intensity. Note that the amplitude of the main field response does not signifcantly change after bicuculline infusion. However, a 1 mV secondary field response (open squares) becomes apparent (see traces shown in E). <b>C.</b> Population data of the main field response amplitude after 10 minutes of bicuculline for 300 and 400 µA stimulation intensity. Values are normalized to baseline. * <i>p</i><0.0001,Wilcoxon paired test. <b>D.</b> Bicuculline consistently increased or induced the appearance of a secondary field response in all recording sites stimulated at 400 µA that showed a field response during baseline condition (32±5%, * <i>p</i><0.001 Wilcoxon paired test). Values are normalized to the main field amplitude during baseline. <b>E.</b> Representative traces showing the effect of intrastriatal bicuculline infusion by reverse microdialysis on local field responses to prelimbic cortex stimulation at 400 µA. Note the increased secondary field response after bicuculline infusion.</p

Evoked field response precedes the peak of multiunitary action potential response.

<p><b>A.</b> Field (3–300 Hz) and action potential (300–3000 Hz) responses at the hot spot to increasing stimulation current intensities. Note the secondary field response (N3 peak, black arrow) at high stimulation intensities (20 trials at each intensity). <b>B.</b> To quantify the action potential response, trials were rectified, smoothed and averaged, allowing the computation of the area and peak latency of the multiunitary action potential response. <b>C.</b> Correlation between the amplitude of the evoked field potential and the area of the action potentials response at 300 µA; each point stems from one recording site at the hot spot from 23 different experiments. r² = 0.84, <i>p</i><0.0001. <b>D.</b> Field response N2 peak precedes the peak of multiunitary action potential response (n = 23, * <i>p</i><0.005, Student's paired t test).</p

Evoked field potential amplitude changes along the dorsal striatum and with stimulation intensity.

<p>A. Representative histological sections showing the location of cortical (arrow, <i>left</i>) stimulation electrode and striatal (st, <i>right</i>) recording electrode. The striatal image was composed by overlaying microphotographs of the same section under transmitted light (tissue) and epifluorescence (electrode, red). Multichannel silicon probes were immersed in a DiI solution before electrophysiological experiments. Traces on the right are local field potentials evoked by stimulating the cortex with different current intensities (individual trials in gray, average in black). Note the higher amplitude of evoked responses at higher stimulation currents. B. Detail of a representative evoked field response. Field potential amplitude was measured between the N2 and P2 peak for each individual trial and then averaged. In these and all further traces positive is upward. Time 0 corresponds to cortical stimulation. C. Topographical reconstruction of stimulation sites (<i>left</i>) and striatal evoked responses (<i>right</i>) in 12 experiments. Focal stimulation (300 µA) at the prelimbic area produces a maximal response in a restricted region of the dorsal striatum conforming a “hot spot” (circle). D. Amplitude of the striatal field response at the hot spot as a function of stimulation current intensity (n = 12 experiments, mean±SD). E. The number of striatal sites that respond to prelimbic cortex stimulation (evoked field potential amplitude higher than 0.3 mV) increases with stimulation intensity. However, many recording sites (38 out of 288 recorded sites) remained unresponsive even at 700 µA.</p

Intrastriatal bicuculline infusion does not block paired pulse facilitation and enhances field response duration.

<p><b>A.</b> Paired pulse ratio (interstimulus interval 50 ms) of the amplitude of the evoked field potentials during baseline and after bicuculline. Top traces: superimposed average traces of the first and second evoked field potentials during baseline and bicuculline condition. Paired pulse facilitation was not blocked by bicuculline indicating that paired pulse facilitation is not due to differential effects of GABAergic neurotransmission during the first and second stimulation pulse. N = 3 experiments. * <i>p</i><0.05 Student's paired t test. <b>B.</b> The overall duration of the striatal field potentials was determined as the time between the first positive peak P1 of the field response and the positive peak of the last supplementary response. During baseline condition, the duration of the first and second evoked potential was comparable. After 10 minutes of bicuculline infusion the overall duration of the first evoked potential was increased 65±7% whereas the second was increased 146±9%.</p