Input: What is the maximum radius of the emitting region for the black body model?

Context:
\section{Introduction}

The publicly available XMM-Newton slew data covers to date around 35\%
of the sky. The soft band (0.2$-$2 keV) sensitivity limit of the slews
(6$\times10^{-13}$\,ergs cm$^{-2}$ s$^{-1}$) is close to that of the
ROSAT All-Sky Survey (RASS; Voges et al.\ 1999), and in the medium
(2$-$12 keV) band, the slew data goes significantly deeper
(4$\times10^{-12}$\,ergs cm$^{-2}$ s$^{-1}$) than all other previous
large area surveys. Over 7700 individual sources have so far been
detected to a positional accuracy of 8\arcsec.  For details on the
the construction and
characteristics of the first released XMM-Newton slew survey
catalogue, see Saxton et al. (2008). For details of the initial
science results from the slew survey, see Read et al. (2006).

The comparison of XMM-Newton slew data with the RASS is now giving,
for the first time, the opportunity to find exotic, extreme
high-variability X-ray bursting objects, e.g. tidal disruption
candidates (Esquej et al. 2007), and also Galactic novae, flare stars,
and flaring white dwarfs, plus eclipsing binaries, AGN and blazars. It
is only with such a large-area survey as the XMM-Newton Slew Survey,
that transient events as these have a chance of being caught.

One such rare event, XMMSL1~J060636.2-694933, which we here show to be
a new Classical Nova, was discovered in an XMM-Newton slew from 18th
July 2006 at a very high count rate of 23.3\,ct s$^{-1}$ (EPIC-pn:
0.2$-$2\,keV).

Classical novae (see Bode \& Evans 2008 for a review) occur in
interacting binary systems consisting of a white dwarf primary star
and a lower-mass secondary star. The nova itself is a cataclysmic
nuclear explosion caused by the accretion of material (via Roche Lobe
overflow or wind accretion) from the secondary star onto the surface
of the white dwarf; here the pressure and temperature at the base of
the accreted material becomes sufficient to trigger a thermonuclear
runaway. A recent review of the thermonuclear processes powering
classical novae can be found in Starrfield et al.\ (2008).  The
accreted material is partially expelled, obscuring the X-ray emission
from the surface of the white dwarf. At later stages, the ejected
material expands further and becomes optically thin, revealing the
nuclear burning on the surface of the white dwarf.  This emission
peaks in the soft X-ray regime and it is known as the super-soft
source (SSS) state (Krautter 2008). Models of the classical nova SSS
state can be found in Tuchman \& Truran (1998) and Sala \& Hernanz
(2005).

Though many classical novae have been observed in X-rays in their SSS
states (Ness et al.\ (2007) for example discuss several examples observed with
Swift), it is in the optical band, early in their outbursts, that
classical novae are almost always discovered. This is because they are
intrinsically optically bright and easily found in inexpensive
wide-area shallow surveys.  XMMSL1~J060636.2-694933 is very unusual
therefore in that it has been discovered, as we shall see, later in
its evolution, in the SSS X-ray state.

In this paper we describe the XMM-Newton slew observations
(Section~2), and the follow-up X-ray observations by the Swift XRT
(Section~3) and XMM-Newton (Section~4). Multiwavelength observations
with Swift-UVOT, Magellan and ASAS are described in Section~5. We then
present a discussion of the results (Section~6), and conclusions.

\begin{table*}[t]
  \caption[]
  {Details of the four XMM-Newton Slew observations and the single (Rev.\,1378)
    dedicated XMM-Newton pointed observation. XMM-Newton revolution, date and observation ID
    are tabulated, together with the 0.2$-$2.0\,keV X-ray properties of XMMSL1~J060636.2-694933;
    position, background-subtracted counts, exposure, count-rate, and detection likelihood. For the
    Rev.\,1378 dedicated observation, these properties are given for all the EPIC cameras combined.
    For the slew observations, only the EPIC-pn values are given. In the first two slews the source
    was not detected, and upper limits are shown in the table.}
  \centering
\begin{tabular}{lccccrrrr}
\hline
Rev  & Date & Obs.\,ID & RA(J2000)   & Dec(J2000) & Counts         & Exposure & Count rate  & Lik. \\
     & (UT) &        &     &            &                & (s) & (s$^{-1}$)  &      \\ \hline
 351 (slew) & 07/11/01 &  9035100003  &        &               & $<$3.6     & 8.8 & $<$0.41 & $<$$\sim$8      \\
 750 (slew) & 12/01/04 &  9075000003  &        &               & $<$3.2     & 17.3 & $<$0.18 & $<$$\sim$8      \\
1210 (slew )& 18/07/06 &  9121000003  & 06:06:36.2 & -69:49:33 & 228.8$\pm$14.1 & 9.8 & 23.4$\pm$1.4 & 1777.1   \\
1246 (slew) & 28/09/06 &  9121460003  & 06:06:36.5 & -69:49:38 &  12.9$\pm$2.4  & 3.4 &  3.8$\pm$0.7 &   54.7   \\
\vspace{-3.5mm}\\
\hline
1378 (pointed) & 19/06/07 &  0510010501  & 06:06:36.5 & -69:49:37 & 1511.0$\pm$44.8 & 8940.0 &  0.20$\pm$0.01 & 4630.4          \\
\hline
\end{tabular}
\label{slewtable}
\end{table*}

\section{XMM-Newton slew observations}

XMMSL1~J060636.2-694933 was discovered in XMM-Newton slew 9121000003
from revolution 1210 on 18th July 2006. Details of the standard
XMM-Newton slew data reduction and analysis used, plus the
source-searching and catalogue cross-correlation etc., are presented
in Saxton et al.  (2008).

The source passed through the EPIC-pn detector in 14\,s, at a small
off-axis angle, such that an effective vignetting-corrected soft band
(0.2$-$2\,keV) exposure time of 9.8\,s was achieved.  A total of 229
source counts lie within a radius of 20\arcsec, yielding a (EPIC-pn:
0.2$-$2\,keV) count rate of 23.4\,ct s$^{-1}$.

The source is seen to have no cross-correlation identifications in the
RASS, and no other multiwavelength candidates within 30\arcsec\ in
Simbad\footnote{http://simbad.u-strasbg.fr/simbad/},
NED\footnote{http://nedwww.ipac.caltech.edu/index.html}, and
HEASARC\footnote{http://heasarc.gsfc.nasa.gov/}. The position of the
source in the sky is such that it lies apparently at the outer eastern
edge of the LMC.

XMM-Newton has slewed over this region of sky a number of times, and
though nothing was detected in previous slews from 7th November 2001
and 12th January 2004, the source was seen again on 28th September
2006 (rev.\,1246, 72 days after the rev.\,1210 discovery), at the same
position, but at a reduced flux level (3.8\,ct s$^{-1}$; EPIC-pn:
0.2$-$2\,keV).  i.e. it had reduced in flux by a factor of $\approx$6
in 72 days. XMM-Newton has not slewed over this area of sky since
rev.\,1246. Details of the relevant XMM-Newton slews, together with
the (0.2$-$2\,keV) EPIC-pn source position, detected source counts,
count rate and detection likelihood are given in
Table~\ref{slewtable}.

The fact that XMMSL1 J060636.2-694933 is detected in the total-band
(0.2$-$12\,keV) and the soft-band (0.2$-$2\,keV), whilst effectively
zero counts are seen in the hard-band (2$-$12\,keV), is immediately
indicative of the source being very soft.

The moderately high count rate indicates that the spectrum is affected
by pile-up (the on-axis limit is 6\,ct s$^{-1}$ for EPIC-pn full-frame
mode
\footnote{http://xmm.esac.esa.int/external/xmm\_user\_support/documentation
  /uhb\_2.5/index.html}). This distorts the spectrum and makes
quantitative spectral analysis of the slew data difficult. We
minimized these effects by following the standard procedure, i.e.
ignoring the central part of the Point Spread Function (PSF), and
extracted an event spectrum (containing single and double events) of
the source from within an annulus of 5\arcsec$-$30\arcsec\ radius,
centred on the source position. Unresolved problems associated with
the motion of sources across the detector still exist within slew
data, and approximations currently have to be made when calculating
the associated effective area and detector response matrix files. In
order to perform qualitative spectral analysis, an effective area file
was generated by averaging the individual core-removed effective area
files at 9 different positions along the detector track made by the
source. This accounts for the removal of the piled-up core, and takes
the vignetting and PSF variations into account to a good
approximation.  Individual BACKSCAL values have been set by hand, as
have the EXPOSURE values, estimated by calculating the distance
travelled by the source in detector coordinates and finding the time
taken to do this, given a 90\,deg\,hr$^{-1}$ slew speed, then
subtracting the appropriate fractions for chip gaps and bad pixels.
For the response matrix, we used the equivalent canned detector
response matrix for the vignetting-weighted average source position,
for single plus double events and for full-frame mode:
epn\_ff20\_sdY6\_v6.9.rmf. A background spectrum was extracted from a
much larger circular region close to the source and at a similar
off-axis angle.

To fit the slew spectral data, and indeed all the high-energy spectra
in the present paper, the
XSPEC\footnote{http://heasarc.gsfc.nasa.gov/docs/xanadu/xspec/}
spectral fitting package has been used. As $\chi^2$ minimization is
not valid when fitting spectra of low statistical quality, for the
fitting of the slew spectrum (and all the spectral fitting in the
present paper), C-statistics have been used. To take into account the
absorbing column along the line of sight, the {\em wabs} model with
the {\em wilm} cosmic abundance table (Wilms et al.\ 2000) has been
used throughout the paper. All the errors quoted in the present paper
are 90\% confidence intervals, unless otherwise stated.

The rev.\,1210 slew spectrum shows that the source is very soft, and
appears consistent with a 63$_{-10}^{+12}$\,eV black body, absorbed by
a hydrogen column density of
8.2$_{-4.1}^{+5.4}\times10^{20}$\,cm$^{-2}$. The fit is good, with a
P-statistic value of 0.11, obtained via the XSPEC {\em goodness}
command for this fit, based on 5000 random simulations. The best-fit
hydrogen column is equal to the full Galactic hydrogen column in the
direction of the source (8.0$\pm{1.1}\times10^{20}$\,cm$^{-2}$; Dickey
\& Lockman, 1990, calculated via the FTOOL {\em
  nh}\footnote{http://heasarc.gsfc.nasa.gov/lheasoft/ftools/fhelp/nh.txt}).
The slew spectrum, plus the best fit simple black body model and the
deviations from the model, are shown in Fig.\,\ref{slewspec}. The
observed count rate corresponds to a (0.2$-$2\,keV) flux, corrected
for the removal of the saturated PSF core, of
4.8$^{+2.7}_{-1.6}\times10^{-11}$\,ergs cm$^{-2}$ s$^{-1}$ (an
increase in flux over the RASS upper limit, assuming the same spectral
model, by a factor of more than 500).

Simple power-law, thermal Bremmstrahlung, and other optically thin hot
plasma models are unable to fit the spectrum adequately well.  Given
that we later are able to identify the source as a nova (Section~5.2),
then the black-body model will likely be a good approximation.
Furthermore, as we have obtained here a moderate number of slew
counts, the more physically realistic, though more complex atmosphere
model for CO white dwarfs of MacDonald \& Vennes (1991), provided by
K.\,Page (private communication), was attempted.  This model, used
e.g. to model the nova V1974 Cyg (Balman et al.\ 1998), yielded a
marginal fit (and not formally a more statistically significant fit;
P-statistic = 0.03, based on 5000 random simulations), with an
effective temperature of 70$^{+8}_{-6}$\,eV, an $N_{\rm H}$ of
3.7$^{+3.2}_{-2.5}$$\times$$10^{20}$\,cm$^{-2}$, and a PSF-corrected
(0.2$-$2\,keV) flux of 4.5$^{+1.3}_{-1.8}\times10^{-11}$\,ergs
cm$^{-2}$ s$^{-1}$. Note that a smaller $N_{\rm H}$ (though perhaps
still consistent with the full Galactic hydrogen column) is now
obtained using the white dwarf atmosphere model. (Note that the
MacDonald \& Vennes (1991) ONe white dwarf atmosphere model was also
attempted, but yielded a marginally worse fit than the CO white dwarf
atmosphere model; only the CO atmosphere model has been used in the
subsequent analysis).

It is well known (e.g. Krautter et al.\ 1996) that, because of the
energy-dependent opacity in the white dwarf atmosphere, fits to super
soft source novae spectra with black body models give larger fluxes
and lower temperatures than atmosphere models fit to the same spectra,
and this is seen in the present case. Thus the black body model
requires a larger $N_{\rm H}$ to fit the same data than the atmosphere
model, as is seen.

The model normalizations, corrected for the removal
of the saturated PSF core, can be used to derive an approximate
distance to the source.  If we assume a typical emitting region for
the white dwarf atmosphere to be of spherical radius 10$^{9}$\,cm,
then, for the black body model, this distance turns out to be
20$^{+31}_{-10}$\,kpc. The effects discussed above however can lead to
usage of the black body model giving rise to an underestimation of the
distance.  For the white dwarf atmosphere model, a larger distance of
71$^{+27}_{-23}$\,kpc is obtained. Both estimates are consistent with
the distance to the LMC ($\sim$50\,kpc, see Section~6), and assuming a
distance of 50\,kpc, the black body derived flux corresponds to a
(pile-up corrected) 0.2$-$2\,keV X-ray luminosity of
1.4$^{+0.8}_{-0.5}\times10^{37}$\,ergs s$^{-1}$.

\begin{figure}
\centering
\includegraphics[bb=100 20 575 700,clip,width=6.0cm,angle=270]{12082f1.ps}
\caption{XMM-Newton Slew spectrum of XMMSL1 J060636.2-694933 from
  XMM-Newton revolution 1210.  The data points (crosses; adjacent data
  bins having been grouped together for the plot to have a significance of at least
  3) have been fitted with a black body model (kT=63\,eV; see text).
  The solid line shows the best fit to the spectrum. The ratio of the
  data to the best fit model is shown in the lower panel.}
\label{slewspec}
\end{figure}

\section{Swift XRT X-ray observations}

We requested and received a prompt observation with Swift of this
source before it moved out of the Swift visibility window in April
2007. We received over 14\,ksec of Swift-XRT time in 7
separate observations and the details of these observations are listed
in Table~\ref{xrttable}. All of the observations were in photon
counting mode and none of the observations showed any times of
significant high-BG flux. In none of the observations did the source
position coincide with any of the dead (micrometeorite-induced)
detector columns. The analysis has been performed using HEASOFT
v6.1.2. The individual XRT observations were astrometrically-corrected
and then stacked to ascertain a best Swift-XRT position $-$ this was
found to be 06 06 37.00 -69 49 33.9 (with a 90\% error radius of
4.0\arcsec). Source counts were then extracted from each observation
from a circle of radius of 40\arcsec\ at this position. Background
counts were extracted from each observation from large-radius
off-source circles close to the source position. Source counts and
count rates for the individual XRT observations are given in
Table~\ref{xrttable}.

\begin{table}
  \caption[]{Details of the Swift-XRT observations (observation ID, observation date and
    cleaned exposure time) are tabulated, together with the total (0.2$-$2.0\,keV) background-subtracted
    counts and count rate from XMMSL1 J060636.2-694933 (see text).}
  \centering
\begin{tabular}{ccrrr}
\hline
ID          & Date     & Exp. & Counts       & Count rate       \\
            &   (UT)   &  (s) &              & (s$^{-1}$)        \\ \hline
00030895001 & 28/02/07 & 1955 & 23.9$\pm$5.1 & 0.0122$\pm$0.0026  \\
00030895002 & 07/03/07 & 1796 & 15.8$\pm$4.2 & 0.0088$\pm$0.0024 \\
00030895003 & 08/03/07 & 1651 & 10.9$\pm$3.6 & 0.0066$\pm$0.0022 \\
00030895004 & 08/03/07 & 2547 & 20.6$\pm$4.8 & 0.0081$\pm$0.0019 \\
00030895005 & 10/03/07 & 2550 & 29.5$\pm$5.7 & 0.0116$\pm$0.0022 \\
00030895006 & 20/03/07 &  552 &  8.6$\pm$3.2 & 0.0156$\pm$0.0057 \\
00030895007 & 22/03/07 & 3391 & 24.4$\pm$5.4 & 0.0072$\pm$0.0016 \\
\hline
\end{tabular}
\label{xrttable}
\end{table}

The observation naturally fell into three time-separated groups, those
of obs.\,1, obs.\,2-5 and obs.\,6-7. A similar analysis applied to
these groups (where the statistics are improved) gives rise to source
counts and count rates of 76.7$\pm$9.3\,counts and
0.0090$\pm$0.0011\,ct~s$^{-1}$ (for obs.\,2-5), and
33.0$\pm$6.2\,counts and 0.0084$\pm$0.0016\,ct~s$^{-1}$ (for
obs.\,6-7). (Analysis of all the data together yields
133.6$\pm$12.3\,counts and 0.0092$\pm$0.0009\,ct~s$^{-1}$).

A spectrum was extracted from all the Swift-XRT data from a 40\arcsec\
radius circle, using grades 0$-$12, centred on the Swift-XRT position.
A background spectrum was extracted again from all the Swift-XRT data,
from large-radius off-source circles close to the source position. An
ARF file was created using {\em xrtmkarf} and the appropriate RMF
(swxpc0to12\_20010101v008.rmf) from the Swift-XRT Calibration Database
was obtained.

Standard spectral models were again fit to the spectral data using
XSPEC. Again, C-statistics were used, as was the {\em wabs} absorption
model with the {\em wilm} cosmic abundance table. It was again
obvious that only a very soft spectrum would be appropriate for the
data, and the only simple model that was able to fit the data
adequately was a black-body model of temperature
$kT$=$59^{+14}_{-10}$\,eV, with an absorbing hydrogen column of
9.5$^{+5.0}_{-3.9}$$\times$$10^{20}$\,cm$^{-2}$. No sufficiently constrained parameters could
be obtained using the CO white dwarf atmosphere model (MacDonald \&
Vennes 1991). The Swift-XRT spectrum, together with the best-fit black
body model is shown in Fig.\,\ref{xrtspec}. The corresponding
(0.2$-$2.0\,keV) flux is 2.7$^{+0.7}_{-1.2}\times10^{-13}$\,ergs
cm$^{-2}$ s$^{-1}$ (i.e. a reduction by more than a factor 100 from
the XMM-Newton slew discovery flux), and the X-ray luminosity, for the
assumed distance of 50\,kpc, is 8.0$^{+2.2}_{-3.5}\times10^{34}$\,ergs
s$^{-1}$.

\begin{figure}
\centering
\includegraphics[bb=100 15 580 710,clip,width=6.0cm,angle=270]{12082f2.ps}
\caption{Swift-XRT spectrum from XMMSL1 J060636.2-694933. The data
  points (crosses; adjacent data bins having been grouped together for
  the plot to have a significance of at least 3) have been fitted with
  a black body model (kT=59\,eV; see text). The source has faded by a
  factor of $>100$ since the XMM-Newton revolution 1210 slew
  discovery. The solid line show the best fit to the spectra.  The
  ratio of the data to the best fit model is shown in the lower panel.
}
\label{xrtspec}
\end{figure}

A cautious estimate of the size of the emitting region can be obtained
from the model normalization; the assumed distance of 50\,kpc yields a
maximum radius of 4.5$\times$10$^{8}$\,cm (the fit normalization is
essentially unconstrained at the lower bound).  Though great care
should be taken in interpreting this result, as the black body model
is possibly overestimating the luminosity, this obtained radius is
still consistent with that of moderately massive ($>$1.1$M_{\odot}$)
white dwarfs (Hamada \& Salpeter 1961), i.e.\,the whole white dwarf
surface may still be emitting at 59\,eV.

\section{Dedicated XMM-Newton observations}

We were granted an XMM-Newton Target of Opportunity (ToO) observation,
once the source became again visible to XMM-Newton, and a 10\,ks
XMM-Newton EPIC observation was made on 19th June 2007 (see
Table~\ref{slewtable}). All the XMM-Newton EPIC data, i.e.  the data
from the two MOS cameras and the single pn camera, were taken in
full-frame mode with the thin filter in place.  These data from the
three EPIC instruments have been reprocessed using the standard
procedures in XMM-Newton SAS (Science Analysis System) $-$ v.7.1.0.
Periods of high-background, of which there were very few, were
filtered out of each dataset by creating a high-energy 10$-$15\,keV
lightcurve of single events over the entire field of view, and
selecting times when this lightcurve peaked above 0.75\,ct s$^{-1}$
(for pn) or 0.25\,ct s$^{-1}$ (for MOS). This resulted in
$\approx$9.4(8.0)\,ks of low-background MOS(pn) data. Details of this dedicated
XMM-Newton observation, together with source position, and
(0.2$-$2\,keV) all-EPIC combined (pn, MOS1, MOS2) detected source
counts, count rate and detection likelihood are given in
Table~\ref{slewtable}.

Source spectra, containing single and double events, were extracted
from the datasets from circles (none of the data were now piled up)
centred on the source position. An extraction radius, estimated from
where the radial surface brightness profile was seen to fall to the
surrounding background level, was set to 30\arcsec. Background spectra
were extracted from each cleaned dataset from a 40\arcsec$-$80\arcsec\
annulus centred on the source position. Point sources seen to
contaminate these larger-area background spectra were removed from the
background spectra to a radius of 60\arcsec. ARF files were created
for the source spectra, and were checked to confirm that the correct
extraction area calculations had been performed. Finally RMF response
files were generated.

Standard spectral models were again fit to the spectral data using
XSPEC. Once again it was obvious that only a very soft model would fit the data; the only
simple model that was able to fit the data well (a P-statistic = 0.17,
based on 5000 random simulations) was a black-body model of
temperature $kT$=70$^{+3}_{-4}$\,eV, with an absorbing hydrogen column
of 6.9$^{+1.0}_{-1.6}\times10^{20}$\,cm$^{-2}$. The spectrum, together with this best-fit
model are shown in Fig.\,\ref{xmmspec}. The corresponding
(0.2$-$2.0\,keV) flux is only marginally less than the Swift-XRT value
at 2.2$^{+0.8}_{-0.9}\times10^{-13}$\,ergs cm$^{-2}$ s$^{-1}$ and the
X-ray luminosity (for the assumed distance of 50\,kpc) is
6.7$^{+2.5}_{-2.8}\times10^{34}$\,ergs s$^{-1}$.

\begin{figure}
\centering
\includegraphics[bb=110 15 570 705,clip,width=6.0cm,angle=270]{12082f3.ps}
\caption{XMM-Newton ToO spectrum from XMMSL1 J060636.2-694933. The
  data points (crosses; adjacent data bins having been grouped
  together for the plot to have a significance of at least 3)) have
  been fitted again with a black body model (kT=70\,eV) (see text).
  EPIC-pn data is shown in black, with EPIC-MOS1 in red and EPIC-MOS2
  in green. The solid lines show the best fit to the spectra.  The
  ratios of the data to the best fit model are shown in the lower
  panel.}
\label{xmmspec}
\end{figure}

Given that, in this XMM-Newton ToO observation, we had obtained a
larger number of counts ($\raisebox{-1mm}{$\stackrel{>}{\sim}$}$1500 over the 3 EPIC cameras), the
physically more realistic CO white dwarf atmosphere model (MacDonald \&
Vennes 1991) was also attempted. This yielded a marginal fit (and formally
a no more statistically significant fit; P-statistic = 0.04, based on
5000 random simulations), with an effective temperature of
73$^{+3}_{-2}$\,eV, and an $N_{\rm H}$ of
3.4$^{+0.8}_{-0.8}$$\times$$10^{20}$\,cm$^{-2}$. Again, usage of the black body model results
in a larger fitted $N_{\rm H}$ and a lower fitted temperature than
with the atmosphere model.

As before, the model normalization can be used to obtain a cautious
estimate of the size of the emitting region. For the assumed distance
of 50\,kpc, then the black body model returns an emitting region
radius of only 1.3$\pm$0.2$\times$10$^{8}$\,cm.  Again care should be
taken, as this may be an overestimation, the black body model having
perhaps overestimated the luminosity. For the white dwarf atmosphere
model, a smaller radius of 0.4$\pm$0.1$\times$10$^{8}$\,cm is
obtained. Note further that the assumption of a larger distance (see
Section~6) would result in a proportionally larger emitting radius.
The range in allowed radius therefore is quite large, and it is not
impossible for for the whole of the white dwarf surface to be emitting
at 70\,eV. If this is the case, then the white dwarf would have to be
at the high end of the mass range ($>$1.2$M_{\odot}$; Hamada \&
Salpeter 1961). It may be the case then that we are at this point at,
or close to the end of the SSS phase, where the effective temperature
has reached a maximum (Sala \& Hernanz 2005), as is tentatively seen
in the spectral fitting results, and where the photospheric radius has
reached a minimum, close to the white dwarf radius.

\subsection{X-ray variability}

The full (XMM-Newton slew plus Swift-XRT plus XMM-Newton ToO) X-ray
lightcurve of XMMSL1 J060636.2-694933 is shown in
Fig.\,\ref{lightcurve}. The calculated (0.2$-$2.0\,keV) flux values
are shown plotted against the number of days since the rev.\,1210
XMM-Newton Slew discovery. The first two data points are the
rev.\,1210 and the rev.\,1246 XMM-Newton Slew observations. Then the
three nested Swift-XRT points are shown and finally the XMM-Newton ToO
observation. The level of RASS upper limit is shown to the bottom
left. The (0.2$-$2.0\,keV) X-ray flux is seen to have dropped by more
than two orders of magnitude in 230 days since the discovery, but is
then seen to have levelled off for the next 120 days, at a level still
$\approx$3 times that of the RASS. Finally, no evidence for any
short-term variability (using time bins down to 100\,s) is seen in the
highest statistic continuous X-ray lightcurve (the $\approx$8.0\,ksec
background-filtered EPIC-pn lightcurve) obtained from the 19/06/07
XMM-Newton observation.

\begin{figure}
\centering
\includegraphics[bb=60 60 550 454,clip,width=8.7cm]{12082f4.ps}
\caption{The full X-ray lightcurve of XMMSL1 J060636.2-694933. Plotted
  are the calculated (0.2$-$2.0\,keV) flux values versus time. The
  first point is the rev.\,1210 XMM-Newton Slew observation, then the
  rev.\,1246 XMM-Newton Slew observation. The three nested Swift-XRT points
  are shown next and finally the XMM-Newton ToO observation. The RASS upper
  limit is shown bottom left. }
\label{lightcurve}
\end{figure}

\section{Multi-wavelength Follow-up}

\subsection{Swift UVOT}

For the Feb/Mar 2007 Swift observations, we arranged for both the
Swift UVOT-B filter and the UVOT-UVW2 filters to be used in an
approximate exposure time ratio of 1:5, thus ensuring roughly equal
numbers of counts in the two bands (though there is a spectral type
dependency here). Swift UVOT images in these two filters of the area
of sky around XMMSL1 J060636.2-694933 are shown in Fig.\,\ref{uvot}.

Prior to the Swift UVOT observations, a `best-guess' to the possible
candidate optical/IR counterpart would have been the USNO-A2.0 source
0150-04066298 (B~mag: 17.4, R~mag: 16.1), seen 4\arcsec\ south of the
XMM-Newton slew position. The UVOT images however immediately showed
that the optically fainter source at position RA, Dec (J2000) = 06 06
36.4, -69 49 34.3 (error radius: ~0.5\arcsec) was a very strong UVW2
source and very blue, and was very likely the true counterpart to
XMMSL1~J060636.2-694933. (The UVW2 filter spans approximately
800\AA\,, centred at $\approx$1900\AA)

\begin{figure}
\centering
\includegraphics[bb=-82 210 695 585,clip,width=8.7cm]{12082f5.ps}
\caption{Swift UVOT images of the field around XMMSL1 J060636.2-694933 from observation
  00030895002. Left shows the UVOT B-filter and right shows the the
  UVOT UVW2-filter. The large circle is a 20\arcsec\ radius circle around
  the XMM-Newton Slew position. The small circle in the UVW2 image around the
  bright source is reproduced in the B image, indicating that a faint
  optical source is also visible at this position.}
\label{uvot}
\end{figure}

The Swift UVOT pipeline processed data were analysed using the UVOT
photometry package {\em uvotsource} released with
FTOOLs\footnote{http://heasarc.nasa.gov/lheasoft/ftools/ftools\_menu.html}.
This package performs aperture photometry on pre-specified source and
background regions, accounting for photometric- (via PSF fitting) and
coincidence loss- effects using the UVOT calibration files. Source
counts were extracted using a 5\arcsec\ radius aperture centred on the
source, while for the background we used a 10\arcsec\ radius aperture
located in a nearby source-free region.  We used a larger background
aperture to effectively smooth over the modulo-8 fixed pattern noise
present in UVOT observations and to improve the statistics of the
background counts. Source counts were converted to UVOT UV-magnitudes
using the UVW2 zero-point calibration released with version~2.8 (Build
22) of the CALDB. The source is seen (see Fig.\,\ref{uvotlc}) to be
roughly constant over the short duration of the Swift observations,
with a suggestion of a decline towards the end. This is in keeping
with the general form of the X-ray lightcurve (Fig.\,\ref{lightcurve})
at this time.

\begin{figure}
\centering
\includegraphics[bb=80 70 535 380,clip,width=8.7cm]{12082f6.ps}
\caption{Variation of the UVW2 magnitude of the bright UV source
  during the Swift observations. The same time axis as
  Fig.\,\ref{lightcurve} has been used to aid comparison, and a zoom
  is also shown. The UVW2 filter was only employed during observations
  00030895002, 00030895004, 00030895005, 00030895006 \& 00030895007
  (hence the points span the dates 07/03/07 to 22/03/07). The errors here are 1-$\sigma$. }
\label{uvotlc}
\end{figure}

It is possible to include the UVOT-detected flux with the XRT spectrum
described in Section~3. UVOT files, created using {\em uvot2pha} for
the five observations (00030895002, 00030895004, 00030895005,
00030895006 \& 00030895007) where the UVW2 filter was employed, were
incorporated into {\em xspec}, along with the appropriate response
file (swuw2\_20041120v104.rsp) from the Swift-XRT Calibration
Database. We attempted to fit a single black-body spectrum to the
Swift-XRT+UV data (again using C-statistics, the {\em wabs} absorption
model and the {\em wilm} cosmic abundance table, plus the inclusion of
the {\em xspec-redden} component to model the absorption in the UV
band). The best fit however, with a much lower temperature of
$kT$=$36^{+3}_{-4}$\,eV, is a very poor fit to the data; we obtain a
{\em goodness} P-statistic value of 0.00, based on 5000 random
simulations. This notwithstanding, a flux in the UVW2
(1.57$-$7.77\,eV) band of 3.5$\pm{0.2}\times10^{-13}$\,ergs cm$^{-2}$
s$^{-1}$ can be obtained, corresponding to a UVW2 luminosity, for the
assumed distance of 50\,kpc, of 1.0$\pm{0.1}\times10^{35}$\,ergs
s$^{-1}$.

The very poor single black-body fit above, plus the large change in
fitted temperature is strongly suggestive that a model other than, or
in addition to the XRT-derived kT=59\,eV black body model (Section~3)
should be used to describe the UVW2 data. As we have no UV data other
than in the UVW2 filter, all that can be done is to apply the
XRT-derived black body model to the UVW2+XRT data, and in doing this,
a large flux excess with respect to the XRT-derived black body model
is seen in the UVW2 band. This is shown in Fig.\ref{xrtuvotspec}. This
excess in UV emission (most of the $10^{35}$\,ergs s$^{-1}$ discussed
above) is likely due to a combination of residual post-nova nuclear
burning on the surface of the white dwarf, plus accretion in the disk,
including from emission lines. The situation is likely to be rather
complex, depending on the structure of both the ejecta and the
accretion disk, and is beyond the scope of the present work, where we
only have sparse UV data.  For a review of the UV emission from
classical novae, see Shore (2008).

\begin{figure}
\centering
\includegraphics[bb=100 15 580 710,clip,width=6.0cm,angle=270]{12082f7.ps}
\caption{Swift-XRT spectrum (black) from XMMSL1 J060636.2-694933, plus
  the best-fit black-body model to this spectrum (Section~3; Fig.\,2),
  but extending into the UV to the Swift-UVOT UVW2 flux points (coloured)
  (see text). The data points are plotted such that adjacent data
  bins have been grouped together to have a significance of at least
  3. The solid line show the best fit to the Swift-XRT spectrum. The
  ratio of the data to the best fit model is shown in the lower
  panel.}
\label{xrtuvotspec}
\end{figure}

\subsection{Magellan optical observations}

On Nov.~13, 14, and 15, 2007, XMMSL1~J060636.2--694933 was observed
with the Low--Dispersion Survey Spectrograph 3 (LDSS3) mounted on the
Magellan Clay telescope. Images were obtained through the Sloan
$g^\prime$, $r^\prime$ and $i^\prime$ filters. On Nov.~15, 2007
conditions were photometric and the Landolt field RU 149A was observed
to flux calibrate the data in the $g^\prime$, $r^\prime$ and
$i^\prime$--bands.  The Landolt (1992) magnitudes of the standards
were converted to Sloan magnitudes using the transformations presented
in Smith et al.\ (2002). All the images were debiased and flatfielded
using dome flatfield frames. We applied aperture photometry on each of
the images using DAOPHOT in \textsc{IRAF}\footnote{\textsc {iraf} is
  distributed by the National Optical Astronomy Observatories} to
compute the instrumental magnitudes of the stars. Differential
photometry of the optical counterpart to XMMSL1~J060636.2-694933
(marked by an arrow in Fig.~\ref{magellan}) was performed with respect
to the field star (marked with a `c' in Fig.~\ref{magellan}). This was the
brightest isolated and unsaturated star common to all frames.  The
calibrated brightness of this comparison star is $g'= 18.42 \pm 0.04$,
$r'= 17.85 \pm 0.06$ and $i'=17.58 \pm 0.07$.

\begin{figure}
\centering
\includegraphics[bb=35 215 575 575,clip,width=8.7cm]{12082f8.ps}
\caption{Magellan Clay LDSS3 finder chart. The counterpart to
  XMMSL1~J060636.2-694933 (and the bright Swift-UVOT UVW2-filter
  source; Figs.\ref{uvot}\&\ref{uvotlc}) is marked with an arrow. The comparison star is
  shown marked with a 'c'.}
\label{magellan}
\end{figure}

In addition to the imaging observations described above, we have
obtained spectroscopic observations on Nov.~13, 14, and 15, 2007 using
the VPH All grism, which has 660 lines per mm, and employing a
1\arcsec\ wide slit. This set-up provides a mean dispersion of 2\AA\,
per pixel. For a slit width of 1 arcsecond and a mean seeing close to
1\arcsec, the mean spectral resolution is $\approx$10\AA. On Nov.~13, 2007
we took 4 exposures of 450\,s each, on Nov.~14, 2007 we took 2
exposures of 900\,s each, and on Nov.~15, 2007 we took one 1200\,s
exposure with the slit at the parallactic angle. The spectra were bias
and flatfield corrected, and extracted in \textsc{IRAF}. The
instrumental response was corrected using the spectrophotometric flux
calibrators LTT 3218 (Nov.~13), H600 (Nov.~14) and LTT 9293 (Nov.~15).
Significant differences in the flux around H$\alpha$ are apparent with
the flux being 50\% higher during the Nov.~15, 2007 with respect to
the Nov.~13, 2007 observations. Since there is no evidence for
brightening in the $r^\prime$ images we attribute the difference to
the fact that the source was not observed at the parallactic angle on
Nov.~13 and 14, 2007. We exported the one dimensional spectra to the
spectral analysis software package \textsc{molly} for further
analysis.

\begin{figure}
\centering
\includegraphics[bb=70 30 600 800,clip,width=6.8cm,angle=270]{12082f9.ps}
\caption{Magellan Clay averaged optical spectrum of the optical source
  associated with XMMSL1 J060636.2-694933. The flux scaling is
  approximate. The prominent strong emission lines are marked (see
  text). }
\label{optspec}
\end{figure}

We have averaged all spectra (see Fig.~\ref{optspec}). We find several
strong emission lines. The strongest of these emission lines are best
interpreted as due to [OIII] 4958.9\AA\, and 5006.9\AA\,, He~II at
4685.8\AA\, and a blend of the H$\alpha$ plus the [NII] at 6548.1\AA\,
and 6583.4\AA\,, lines found often in novae (Williams 1992). In this
case the main [OIII] lines appear redshifted by approximately 2000\,km
s$^{-1}$. We interprete this as due to clumpy outflows in the nova
shell. The integrated light from different outflowing parts can also
explain the substructure that is present in the [OIII] lines. The
outflow velocities that we obtain for the H$\alpha$ and H$\beta$ lines
is $\approx$350\,km s$^{-1}$, hence less than that for the [OIII]
lines.  Note that, if XMMSL1~J060636.2-694933 does reside within the
LMC, then the systematic line-of-sight recession velocity of the LMC,
262$\pm$3.4\,km~s$^{-1}$ (van der Marel et al.\ 2002), should be taken
into account; i.e.\,a good fraction of the observed H$\alpha$ and H$\beta$
recession would then be due to the recession of the LMC itself.

\subsection{Long-term Optical light curve}

Analysis of archival robotic optical survey data from 3-minute CCD
exposures (pixel size 14\arcsec.8), obtained with a 70\,mm (200\,mm
focal length) f/2.8 telephoto lens in the course of the All Sky
Automated Survey (ASAS; Pojmanski 2002) show that the visual magnitude
of this source rose from m$_{V}\raisebox{-1mm}{$\stackrel{>}{\sim}$}$14 to m$_{V}$$\approx$12 between
Sep.~18, 2005 and Sep.~30, 2005, and then declined rapidly thereafter (see
Fig.\ref{optlc}). ASAS did not detect any significant emission from
the source after around November 2005, the source having dimmed below
the limiting magnitude of ASAS.

The decline from the brightest data point ($\approx$2.2 magnitudes in
10 days, then a further $\sim$1.3 magnitudes in 46 days) suggests that
this is a nova of the 'very fast' speed class (Warner 1995, Downes
et al.\ 2001). We estimate that the time that the light curve takes to
decline 2 magnitudes below maximum observed brightness is
8$\pm$2\,days (see Section~6).

\begin{figure}
\centering
\includegraphics[bb=30 78 453 549,clip,width=7.8cm,angle=270]{12082f10.ps}
\caption{All Sky Automated Survey V-band magnitudes of the optical counterpart
to XMMSL1~J060636.2-694933, during outburst (late September 2005) and afterwards.}
\label{optlc}
\end{figure}

\section{Discussion}

The optical spectrum, showing lines of [OIII] 4958.9\AA\, and
5006.9\AA\,, He~II at 4685.8\AA\, and a blend of the H$\alpha$ plus
[NII] at 6548.1\AA\, and 6583.4\AA\, suggests that
XMMSL1~J060636.2-694933 was a nova, observed (in Nov 2007) in the late
A$_{0}$ auroral phase. The fact that the observed [OIII] lines are not
in the more usual, optically thin 3:1 ratio, can be explained in terms
of a clumpy outflow scenario, whereby individual clumps of both
rest-frame and redward-shifted material are observed, and the
superposition of these account for the observed [OIII] ratio (note
further that density enhancements can change observed [OIII] ratios to
more like $\sim$1:1). Clumps of material are often seen in nova ejecta
(e.g. Shara et al.\ 1997), and outflows of speeds around 2000\,km
s$^{-1}$ are not uncommon in novae (e.g. in nova LMC 1991; Schwartz
et al.\ 2001).

XMMSL1~J060636.2-694933 was likely at its onset (in Oct 2005) a very
fast, Fe~{\sc ii} nova (Section~3 and Williams et al.\ 1991; Williams
et al.\ 1994). An accurate classification now however is not possible,
so late after maximum brightness. The soft ($kT_{\rm
  eff}$$\approx$60--70\,eV) X-ray spectrum indicates that the nova was
in a super-soft source (SSS) state (Krautter 2008) during its
discovery (in July 2006), and throughout its X-ray decline (by more
than two orders of magnitude) in the observations of Sept 2006, March
2007 and June 2007.  Such a state originates from nuclear burning on
the surface of the white dwarf, and measurements of the intensity,
duration, and temperature can be used to estimate the distance to the
nova and the mass of the white dwarf (e.g. Balman et al.\ 1998; Lanz
et al.\ 2005). Indeed, we believe (Section~4) that the white dwarf
within XMMSL1~J060636.2-694933 may be quite massive
($>$1.2$M_{\odot}$).

As discussed earlier, classical novae are almost always discovered
optically in the early phases of their outbursts.
XMMSL1~J060636.2-694933 is very unusual therefore in that it has been
discovered first in X-rays. As such, it is useful to compare it with
XMMSL1~J070542.7-381442 (also known as V598 Pup; Read et al.\ 2008),
another nova recently discovered (in X-rays) in the XMM-Newton slew
survey. With a peak $m_{V}$ of $\ltsim12$, XMMSL1~J060636.2-694933 is
not a particularly bright nova (c.f. V598 Pup, which reached an
m$_{V}$ of $\raisebox{-1mm}{$\stackrel{<}{\sim}$}$4), and so it is not surprising that it went
unnoticed, only being discovered in X-rays during the later (here
291\,days after the outburst), optically thin nebular phase, when
classical novae are typically observed as soft X-ray sources. Though
this delay should be taken as a upper limit, it is long when compared
to V598 Pup ($\raisebox{-1mm}{$\stackrel{<}{\sim}$}$127 days), but may instead be more similar to the
delays of $\sim$200 days seen in V1974 Cyg (Krautter et al.\ 1996),
$\sim$6 months of V382 Vel (Orio et al.\ 2002), and 6$-$8 months of
V1494 Aql (Drake et al.\ 2003). In their X-ray monitoring of optical
novae in M31, Pietsch et al.\ (2007) detect 11 out of 34 novae in
X-rays within a year after their optical outbursts. Seven novae are
seen to be X-ray bright, several (3$-$9) years after outburst, and
three novae showed very short X-ray outbursts, starting within
50\,days of outburst, but lasting only two to three months.
XMMSL1~J060636.2-694933 therefore is not particularly unusual.

A method to estimate the distance to the nova is to use the relation
between the absolute magnitude at maximum brightness and the time that
the light curve takes to decline 2 magnitudes below maximum
brightness, $t_{2}$ (Della Valle \& Livio 1995). We have no
information over the 12 days between the data point of maximum
brightness and the lower limit prior to this (Fig.\,\ref{optlc}), and
therefore we have no exact outburst date, nor exact apparent
magnitude at outburst.  Assuming for the moment though that we have
caught the outburst exactly in the Sep.~30, 2005 observation, then we
can estimate (Sect.~5.3) $t_{2}$ to be 8$\pm$2\,days, and using this,
we can estimate (Della Valle \& Livio 1995) the absolute magnitude at
maximum brightness $M_{V}$ to be --8.7$\pm$0.6. An absolute magnitude
of $M_{V}$=--8.7 implies a peak luminosity $\sim$7 times the Eddington
luminosity for a 1\,$M_{\odot}$ white dwarf. This is quite typical of
novae.

With $A_{V}$=0.39$^{+0.05}_{-0.09}$ (90\% error), as derived (Predehl
\& Schmitt 1995) from $N_{\rm
  H}$=6.9$^{+1.0}_{-1.6}\times10^{20}$\,cm$^{-2}$ (from the highest
statistic spectral fit; the XMM-Newton ToO observation), and with
$M_{V}$=--8.7$\pm$0.6, and a peak $m_{V}$ of 12.0, we can derive a
distance to XMMSL1~J060636.2-694933 of 115$^{+43}_{-30}$\,kpc.  As
discussed above however, we are unsure as to the exact outburst date
and the maximum brightness at outburst. Our assumed peak $m_{V}$ of
12.0 is almost certainly an underestimation. Although we have no
information in the 12 days prior to Sep.~30, 2005, a simple linear
extrapolation of the early October lightcurve back prior to Sep.~30,
2005 suggests that the actual peak $m_{V}$ was somewhere between 9 and
12. The corresponding distance estimates are then between 29 and
115\,kpc (with a mid-point $m_{V}$=10.5 value yielding a distance
estimate of 58\,kpc). Many methods have been used to estimate the
distance to the LMC (e.g.  Kovacs 2000, Nelson et al.\ 2000), but a
value of around 50\,kpc appears to be quite robust. Our distance
estimate is certainly consistent with that of the LMC, though the
errors are quite large.  It does appear to be the case however, that
our distance estimate places the source far outside of our own Galaxy.
This, together with the source's position on the sky (at the eastern
edge of the LMC) and the sizable ($\sim$Galactic) X-ray hydrogen
column densities obtained from the spectral fits, suggest strongly
that XMMSL1~J060636.2-694933 lies within the LMC itself.  Note further
that the (pile-up corrected) spectral model normalizations to the
initial Slew discovery data (Sect.~2) also imply an approximate
distance to XMMSL1~J060636.2-694933 of $\sim$50\,kpc.

The source had, at the time of the slew detection, an absorbed
(0.2$-$2\,keV) X-ray flux of 4.8$^{+2.7}_{-1.6}\times10^{-11}$\,ergs
cm$^{-2}$ s$^{-1}$, corresponding to a 0.2$-$2\,keV X-ray luminosity
(at 50\,kpc) of 1.4$^{+0.8}_{-0.5}\times10^{37}$\,ergs s$^{-1}$.
Assuming instead for the moment a distance more like 100\,kpc (though
this is thought to be well beyond the LMC, e.g. Kovacs 2000), then the
(0.2$-$2\,keV) X-ray luminosity of
5.7$^{+3.0}_{-1.9}\times$$10^{37}$\,erg s$^{-1}$ obtained is at the high end of the X-ray luminosities of
classical SSS-phase novae discussed e.g.\,in Orio et al.\ (2002) and
Ness et al.\ (2007). As discussed though, we have very likely missed
the outburst peak, and as such, our more probable assumed distance of
50\,kpc gives rise to a more typical SSS-phase X-ray luminosity.  The
luminosities of 7$-$8$\times$$10^{34}$\,erg s$^{-1}$, obtained during
the Swift and pointed XMM-Newton observations, are more typical of
novae at later times, when the emission can also sometimes be
described by a thermal plasma, rather than a black-body type spectrum,
or a more mixed spectrum, due to the complex structure of the ejecta
and the accretion disk (Krautter 2008, Shore 2008).

\section{Conclusions}

A bright X-ray source, XMMSL1~J060636.2-694933, was detected in an
XMM-Newton slew on 18 July 2006 at a position where no previous X-ray
source had been seen. The XMM-Newton slew data, plus follow-up dedicated
XMM-Newton and Swift observations, plus optical imaging and
spectroscopic data acquired with the Magellan Clay telescope and
All-Sky Automated Survey (ASAS) data were used to classify the new object
as a nova, and to examine its properties. The primary conclusions are
as follows:

  \begin{itemize}

  \item The soft X-ray spectrum indicates that the nova was in a
    super-soft source (SSS) state at its discovery in July 2007
    (XMM-Newton slew) and through its X-ray decline (by over two
    orders of magnitude) in September 2006 (XMM-Newton slew), March
    2007 (Swift) and June 2007 (XMM-Newton).

  \item The Magellan optical spectrum (Nov 2007) of the source
    indicates that it was very likely then a nova in the late
    A$_{0}$ auroral phase.

  \item The very fast optical decline (ASAS) during the nova's onset
    (Oct 2005), indicates that the initial nova was likely of speed class
    'very fast'.

  \item The very fast speed, together with the absolute magnitude at
    maximum brightness and the X-ray absorption, give rise to a
    distance to the source far beyond our own Galaxy. The large
    distance, together with the source's position in the sky, at the
    eastern edge of the LMC, and the spectral information from the
    X-ray data, are very suggestive that the nova is situated within
    the LMC itself.

  \item Analysis of XMM-Newton slew data is continuing to provide a
    powerful means of finding new X-ray transient objects.

\end{itemize}

\begin{acknowledgements}

  The XMM-Newton project is an ESA Science Mission with instruments
  and contributions directly funded by ESA Member States and the USA
  (NASA). The XMM-Newton project is supported by the Bundesministerium
  f\"ur Wirtschaft und Technologie/Deutsches Zentrum f\"ur Luft- und
  Raumfahrt (BMWI/DLR, FKZ 50 OX 0001), the Max-Planck Society and the
  Heidenhain-Stiftung. AMR and PE acknowledge the support of STFC
  funding, and PGJ of the Netherlands Organisation for Scientific
  Research. The ASAS project is supported by the N2030731/1328 grant
  from the MNiSzW. We thank the referee (G.\,Sala) for very useful
  comments and several references that have improved the paper
  notably. We thank Kim Page for providing the white dwarf atmosphere
  model, and we thank her and Graham Wynn for useful discussions. The
  use of the spectral analysis software package \textsc{molly} written
  by Tom Marsh is also acknowledged. MM acknowledges support by a
  Miller Institute Research Fellowship during the time in which part
  of the work was completed.

\end{acknowledgements}

Answer: 4.5x10^8 cm.


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Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.Neuroscience, Issue 87, Dendritic Spine, Microscopy, Confocal, Fluorescence, Neurosciences, hippocampus, primary neuron, super resolution microscopy, structured illumination microscopy (SIM), neuroscience, dendrite51276Play ButtonSetting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated TransportAuthors: Yves Molino, Françoise Jabès, Emmanuelle Lacassagne, Nicolas Gaudin, Michel Khrestchatisky. Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),51278Play ButtonInducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing RatesAuthors: Alison X. Xie, Kelli Lauderdale, Thomas Murphy, Timothy L. Myers, Todd A. Fiacco. Institutions: University of California Riverside, University of California Riverside, University of California Riverside.Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse51458Play ButtonInhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA ReceptorsAuthors: Laura E. Brown, Celine Fuchs, Martin W. Nicholson, F. Anne Stephenson, Alex M. Thomson, Jasmina N. Jovanovic. Institutions: University College London.Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts. Neuroscience, Issue 93, Developmental neuroscience, synaptogenesis, synaptic inhibition, co-culture, stable cell lines, GABAergic, medium spiny neurons, HEK 293 cell line52115Play ButtonTwo-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull PreparationAuthors: Xinzhu Yu, Yi Zuo. Institutions: University of California, Santa Cruz.In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.Neuroscience, Issue 87, dendritic spine, mouse cortex, in vivo, two-photon microscopy, thinned-skull, imaging51520Play ButtonModeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered MiceAuthors: Robert S. McNeill, Ralf S. Schmid, Ryan E. Bash, Mark Vitucci, Kristen K. White, Andrea M. Werneke, Brian H. Constance, Byron Huff, C. Ryan Miller. Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft51763Play ButtonPaired Whole Cell Recordings in Organotypic Hippocampal SlicesAuthors: Chantelle Fourie, Marianna Kiraly, Daniel V. Madison, Johanna M. Montgomery. Institutions: University of Auckland, Stanford University.Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.Neuroscience, Issue 91, hippocampus, paired recording, whole cell recording, organotypic slice, synapse, synaptic transmission, synaptic plasticity51958Play ButtonImaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium IndicatorsAuthors: Ruotian Jiang, Martin D. Haustein, Michael V. Sofroniew, Baljit S. Khakh. Institutions: University of California Los Angeles, University of California Los Angeles.Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.Neuroscience, Issue 93, astrocyte, calcium, striatum, GECI, GCaMP3, AAV2/5, stereotaxic injection, brain slice, imaging51972Play ButtonMethods to Assess Subcellular Compartments of Muscle in C. elegansAuthors: Christopher J. Gaffney, Joseph J. Bass, Thomas F. Barratt, Nathaniel J. Szewczyk. Institutions: University of Nottingham.Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.Developmental Biology, Issue 93, Physiology, C. elegans, muscle, mitochondria, sarcomeres, ageing52043Play ButtonImproved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term PotentiationAuthors: Agnès Villers, Laurence Ris. Institutions: University of Mons.Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.Neuroscience, Issue 76, Neurobiology, Anatomy, Physiology, Biomedical Engineering, Surgery, Memory Disorders, Learning, Memory, Neurosciences, Neurophysiology, hippocampus, long-term potentiation, mice, acute slices, synaptic plasticity, in vitro, electrophysiology, animal model50483Play ButtonIn Vivo Modeling of the Morbid Human Genome using Danio rerioAuthors: Adrienne R. Niederriter, Erica E. Davis, Christelle Golzio, Edwin C. Oh, I-Chun Tsai, Nicholas Katsanis. Institutions: Duke University Medical Center, Duke University, Duke University Medical Center.Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others. Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model50338Play ButtonDirect Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)Authors: Samira Samtleben, Juliane Jaepel, Caroline Fecher, Thomas Andreska, Markus Rehberg, Robert Blum. Institutions: University of Wuerzburg, Max Planck Institute of Neurobiology, Martinsried, Ludwig-Maximilians University of Munich.Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.Cellular Biology, Issue 75, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging50317Play ButtonPreparation of Dissociated Mouse Cortical Neuron CulturesAuthors: Lutz G. W. Hilgenberg, Martin A. Smith. Institutions: University of California, Irvine (UCI).This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.Neuroscience, Issue 10, cellular, molecular, neurobiology, neuron, calcium/sodium imaging, primary cultures, mouse562Play ButtonAnalysis of Schwann-astrocyte Interactions Using In Vitro AssaysAuthors: Fardad T. Afshari, Jessica C. Kwok, James W. Fawcett. Institutions: University of Cambridge.Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.Cellular Biology, Issue 47, Schwann cell, astrocyte, boundary, migration, repulsion2214Play ButtonQuantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse NumberAuthors: Dominic M. Ippolito, Cagla Eroglu. Institutions: Duke University, Duke University.One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.Neuroscience, Issue 45, synapse, immunocytochemistry, brain, neuron, astrocyte2270Play ButtonPreparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid PathologyAuthors: Diana M. Mathis, Jennifer L. Furman, Christopher M. Norris. Institutions: University of Kentucky College of Public Health, University of Kentucky College of Medicine, University of Kentucky College of Medicine.The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.Neuroscience, Issue 49, aging, amyloid, hippocampal slice, synaptic plasticity, Ca2+, CA1, electrophysiology2330Play ButtonMesenteric Artery Contraction and Relaxation Studies Using Automated Wire MyographyAuthors: Lakeesha E. Bridges, Cicely L. Williams, Mildred A. Pointer, Emmanuel M. Awumey. Institutions: North Carolina Central University, Durham, North Carolina Central University, Durham, Wake Forest University School of Medicine.Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 μm in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 μm. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
Medicine, Issue 55, cardiovascular, resistant arteries, contraction, relaxation, myography3119Play ButtonVisualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero ElectroporationAuthors: Emilie Pacary, Matilda A. Haas, Hendrik Wildner, Roberta Azzarelli, Donald M. Bell, Djoher Nora Abrous, François Guillemot. Institutions: MRC National Institute for Medical Research, National Institute for Medical Research, Université de Bordeaux.In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.Neuroscience, Issue 65, Developmental Biology, Molecular Biology, Neuronal development, In utero electroporation, dendrite, spines, hippocampus, cerebral cortex, gain and loss of function4163Play ButtonImaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture SystemAuthors: Haruki Higashimori, Yongjie Yang. Institutions: Tufts University, Tufts Sackler School of Graduate Biomedical Sciences.Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.Neuroscience, Issue 68, Molecular Biology, Cellular Biology, Biophysics, Microfluidics, Microfluidic culture platform, Compartmented culture, Neuron to glia signaling, neurons, glia, cell culture4448Play ButtonFluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal NeuronsAuthors: Chan-Ying Zheng, Ronald S. Petralia, Ya-Xian Wang, Bechara Kachar. Institutions: National Institutes of Health, Bethesda.FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.Neuroscience, Issue 50, Spine, FRAP, hippocampal neurons, live cell imaging, protein mobility2568Play ButtonPrimary Neuronal Cultures from the Brains of Late Stage Drosophila PupaeAuthors: Beatriz Sicaeros, Jorge M. Campusano, Diane K. O'Dowd. Institutions: University of California, Irvine (UCI).In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

Answer: They are rich in actin and have been shown to be highly dynamic.