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# A negative answer to Nevanlinna’S Type question and a parabolic surface with a lot of negative curvature

## 1. Introduction

The uniformization theorem states that for every simply-connected Riemann surface $`X`$ there exists a conformal map $`\phi :X_0X`$, where $`X_0`$ is either the complex plane $``$, the open unit disc $`𝔻=\{z:|z|<1\}`$, or the extended complex plane (Riemann sphere) $`\overline{}=\{\mathrm{}\}`$, and these possibilities are mutually exclusive . The map $`\phi `$ is called the *uniformizing map*. A simply-connected Riemann surface $`X`$ is said to have *hyperbolic*, *parabolic*, or *elliptic* *type*, according to whether it is conformally equivalent to $`𝔻`$, $``$, or $`\overline{}`$, respectively. In what follows, we assume that $`X`$ is not compact, thus excluding the elliptic case from consideration.

We are interested in the application of the Uniformization Theorem to the following construction. A *surface spread over the sphere* is a pair $`(X,\psi )`$, where $`X`$ is a topological surface and $`\psi :X\overline{}`$ a continuous, open and discrete map. The map $`\psi `$ is called a *projection*. Two such surfaces $`(X_1,\psi _1),(X_2,\psi _2)`$ are *equivalent*, if there exists a homeomorphism $`\varphi :X_1X_2`$, such that $`\psi _1=\psi _2\varphi `$. According to a theorem of Stoïlow , a continuous open and discrete map $`\psi `$ near each point $`z_0`$ is homeomorphically equivalent to a map $`zz^k`$. The number $`k=k(z_0)`$ is called the *local degree* of $`\psi `$ at $`z_0`$. If $`k1`$, $`z_0`$ is called a *critical point* and $`\psi (z_0)`$ a *critical value*. The set of critical points is a discrete subset of $`X`$. The theorem of Stoïlow implies that there exists a unique conformal structure on $`X`$ which makes $`\psi `$ into a meromorphic function. If $`X`$ is simply-connected, what is the type of the Riemann surface so obtained? This is one version of the type problem. Equivalent surfaces have the same type.

Rolf Nevanlinna’s problem concerns a particular class of surfaces spread over the sphere, denoted by $`F_q`$. Let $`\{a_1,\mathrm{},a_q\}`$ be distinct points in $`\overline{}`$.

###### Definition.

A surface $`(X,\psi )`$ belongs to the class $`F_q=F(a_1,\mathrm{},a_q)`$, if $`\psi `$ restricted to the complement of $`\psi ^1\left(\{a_1,\mathrm{},a_q\}\right)`$ is a covering map onto its image $`\overline{}\{a_1,\mathrm{},a_q\}`$.

Assume that $`(X,\psi )F_q`$ and $`X`$ is noncompact. We fix a Jordan curve $`L`$, visiting the points $`a_1,\mathrm{},a_q`$ in cyclic order. The curve $`L`$ is usually called a *base curve*. It decomposes the sphere into two simply-connected regions $`H_1`$, the region to the left of $`L`$, and $`H_2`$, the region to the right of $`L`$. Let $`L_i`$, $`i=1,2,\mathrm{},q`$, be the arc of $`L`$ from $`a_i`$ to $`a_{i+1}`$ (with indices taken modulo $`q`$). Let us fix points $`p_1`$ in $`H_1`$ and $`p_2`$ in $`H_2`$, and choose $`q`$ Jordan arcs $`\gamma _1,\mathrm{},\gamma _q`$ in $`\overline{}`$, such that each arc $`\gamma _i`$ has $`p_1`$ and $`p_2`$ as its endpoints, and has a unique point of intersection with $`L`$, which is in $`L_i`$. We take these arcs to be interiorwise disjoint, that is, $`\gamma _i\gamma _j=\{p_1,p_2\}`$ when $`ij`$. Let $`\mathrm{\Gamma }^{}`$ denote the graph embedded in $`\overline{}`$, whose vertices are $`p_1,p_2`$, and whose edges are $`\gamma _i,i=1,\mathrm{},q`$, and let $`\mathrm{\Gamma }=\psi ^1(\mathrm{\Gamma }^{})`$. We identify $`\mathrm{\Gamma }`$ with its image in $`^2`$ under an orientation preserving homeomorphism of $`X`$ onto $`^2`$. The graph $`\mathrm{\Gamma }`$ has the following properties:

1. $`\mathrm{\Gamma }`$ is infinite, connected;
2. $`\mathrm{\Gamma }`$ is homogeneous of degree $`q`$;
3. $`\mathrm{\Gamma }`$ is bipartite.

A graph, properly embedded in the plane and satisfying these properties is called a *Speiser graph*, also known as a *line complex*. The vertices of a Speiser graph $`\mathrm{\Gamma }`$ are traditionally marked by $`\times `$ and $``$, such that each edge of $`\mathrm{\Gamma }`$ connects a vertex marked $`\times `$ with a vertex marked $``$. Such a marking exists, since $`\mathrm{\Gamma }`$ is bipartite. Each *face* of $`\mathrm{\Gamma }`$, i.e., connected component of $`^2\mathrm{\Gamma }`$, has either a finite even number of edges along its boundary, in which case it is called an *algebraic elementary region*, or infinitely many edges, in which case it is called a *logarithmic elementary region*. Two Speiser graphs $`\mathrm{\Gamma }_1,\mathrm{\Gamma }_2`$ are said to be *equivalent*, if there is a sense-preserving homeomorphism of the plane, which takes $`\mathrm{\Gamma }_1`$ to $`\mathrm{\Gamma }_2`$.

The above construction is reversible. Suppose that the faces of a Speiser graph $`\mathrm{\Gamma }`$ are labelled by $`a_1,\mathrm{},a_q`$, so that when going counterclockwise around a vertex $`\times `$, the indices are encountered in their cyclic order, and around $``$ in the reversed cyclic order. We fix a simple closed curve $`L\overline{}`$ passing through $`a_1,\mathrm{},a_q`$. Let $`H_1,H_2,L_1,\mathrm{},L_q`$ be as before. Let $`\mathrm{\Gamma }^{}`$ be the planar dual of $`\mathrm{\Gamma }`$. If $`e`$ is an edge of $`\mathrm{\Gamma }^{}`$ from a face of $`\mathrm{\Gamma }`$ marked $`a_j`$ to a face of $`\mathrm{\Gamma }`$ marked $`a_{j+1}`$, let $`\psi `$ map $`e`$ homemorphically onto the corresponding arc $`L_j`$ of $`L`$. This defines $`\psi `$ on the edges and vertices of $`\mathrm{\Gamma }^{}`$. We then extend $`\psi `$ to the faces of $`\mathrm{\Gamma }^{}`$ in the obvious way. This defines a surface spread over the sphere $`(^2,\psi )F(a_1,\mathrm{},a_q)`$. See  for further details.

For a Speiser graph $`\mathrm{\Gamma }`$, Nevanlinna introduces the following characteristics. Let $`V(\mathrm{\Gamma })`$ denote the set of vertices of the graph $`\mathrm{\Gamma }`$. To each vertex $`vV(\mathrm{\Gamma })`$ we assign the number

$$E(v)=2\underset{fF(v)}{}(11/k_f),$$

where $`F(v)`$ denotes the set of faces containing $`v`$ on their boundary and $`2k_f`$ is the number of edges on the boundary of $`f`$, $`k_f\{1,2,\mathrm{},\mathrm{}\}`$. The function $`E:V(\mathrm{\Gamma })`$ is called the *excess*. This definition is motivated by considering the curvature, as follows. The $`\psi `$-pullback of the spherical metric $`2|dw|/(1+|w|^2)`$ is generally singular, i.e., it may degenerate on $`\psi ^1(\{a_1,\mathrm{},a_q\})`$. The surface $`X`$, endowed with the pullback metric, is a spherical polyhedral surface, which is a particular kind of orbifold. The *integral curvature* $`\omega `$ on $`X`$ is a signed Borel measure, so that for each Borel subset $`BX`$, $`\omega (B)`$ is the area of $`B`$ with respect to the pullback metric minus $`2\pi _z(k_z1)`$, where the sum is over all critical points $`zB`$ and $`k_z`$ is the local degree of $`\psi `$ at $`z`$.

Each vertex of $`\mathrm{\Gamma }`$ represents a hemisphere, and each face of $`\mathrm{\Gamma }`$ with $`2k`$ edges, represents a critical point, where $`k`$ is the local degree of $`f`$ at this point. Therefore, each vertex of $`\mathrm{\Gamma }`$ has positive integral curvature $`2\pi `$, and each face with $`2k`$ edges has negative integral curvature $`2\pi (k1)`$. We assign the negative curvature evenly to all the vertices of the face. A face with infinitely many edges contributes $`2\pi `$ to each vertex on its boundary. The curvature assigned to every $`vV(\mathrm{\Gamma })`$ is exactly $`2\pi E(v)`$.

Nevanlinna also defines the mean excess of a Speiser graph $`\mathrm{\Gamma }`$. We fix a base vertex $`v_0V(\mathrm{\Gamma })`$, and consider an exhaustion of $`\mathrm{\Gamma }`$ by a sequence of finite graphs $`\mathrm{\Gamma }_{(i)}`$, where $`\mathrm{\Gamma }_{(i)}`$ is the ball of combinatorial radius $`i`$, centered at $`v`$. By averaging $`E`$ over all the vertices of $`\mathrm{\Gamma }_{(i)}`$, and taking the limit, we obtain the *mean excess*, denoted $`E_m=E_m(\mathrm{\Gamma })`$, provided that the limit exists. If the limit does not exist, we consider the upper mean excess $`\overline{E}_m`$ and lower mean excess $`\underset{¯}{E}_m`$, which are the upper ($`lim\; sup`$) and lower ($`lim\; inf`$) limits, respectively.

Nevanlinna’s Problem . Does $`\underset{¯}{E}_m0`$ imply that the surface $`X`$ with the pullback complex structure is parabolic? Conversely, does $`\overline{E}_m<0`$ imply that it is hyperbolic?

Teichmüller  constructed an example of a surface with hyperbolic type, for which the mean excess is zero, thus giving a negative answer to the first question.

We will shortly prove that the answer to the other question is negative as well, by constructing a parabolic surface $`(^2,\psi )F_3`$ with $`E_m<0`$.

In Section 3, we shall construct an example of a non-positively curved, simply-connected, complete, parabolic surface, whose curvature in any ball about a fixed basepoint is less than a negative constant times the area of the ball.

## 2. Counterexample

P. Doyle  proved that the surface $`(X,\psi )`$ is parabolic if and only if a certain modification of the Speiser graph is recurrent. (See  and  for background on recurrence and transience of infinite graphs.) In the particular case where $`k_f`$ is bounded, the recurrence of the Speiser graph itself is equivalent to $`(X,\psi )`$ being parabolic. Though we will not really need this fact, it is not too hard to see that in a Speiser graph satisfying $`E_m<0`$ the number of vertices in a ball grows exponentially with the radius. Thus, we may begin searching for a counterexample by considering recurrent graphs with exponential growth. A very simple standard example of this sort is a tree constructed as follows. In an infinite $`3`$-regular tree $`T_3`$, let $`v_0,v_1,\mathrm{}`$ be an infinite simple path. Let $`T`$ be the set of vertices $`u`$ in $`T_3`$ such that $`d(u,v_n)n`$ for all sufficiently large $`n`$. Note that there is a unique infinite simple path in $`T`$ starting from any vertex $`u`$. This implies that $`T`$ is recurrent. It is straightforward to check that the number of vertices of $`T`$ in the ball $`B(v_0,r)`$ grows exponentially with $`r`$.

Our Speiser graph counterexample is a simple construction based on the tree $`T`$. Fix a parameter $`s\{1,2,\mathrm{}\}`$, whose choice will be discussed later. To every leaf (degree one vertex) $`v`$ of $`T`$ associate a closed disk $`S(v)`$ and on it draw the graph indicated in Figure 1.(a), where the number of concentric circles, excluding $`S(v)`$, is $`s`$. If $`v`$ is not a leaf, then it has degree $`3`$. We then associate to it the graph indicated in Figure 1.(b), drawn on a triply connected domain $`S(v)`$. We combine these to form the Speiser graph $`\mathrm{\Gamma }`$ as indicated in figure 2, by pasting the outer boundary of the surface corresponding to each vertex into the appropriate inner boundary component of its parent. Here, the parent of $`v`$ is the vertex $`v^{}`$ such that $`d(v^{},v_n)=d(v,v_n)1`$ for all sufficiently large $`n`$.

Every vertex of $`\mathrm{\Gamma }`$ has degree $`4`$ and every face has $`2`$, $`4`$ or $`6`$ edges on its boundary. Therefore, $`\mathrm{\Gamma }`$ is a Speiser graph. Consequently, as discussed above, there is a surface spread over the sphere $`X=(^2,\psi )`$ whose Speiser graph is $`\mathrm{\Gamma }`$. It is immediate to verify that $`\mathrm{\Gamma }`$ is recurrent, for example, by the Nash-Williams criterion. Doyle’s Theorem  then implies that $`X`$ is parabolic. Alternatively, one can arrive at the same conclusion by noting that there is an infinite sequence of disjoint isomorphic annuli on $`(^2,\mathrm{\Gamma })`$ separating any fixed point from $`\mathrm{}`$, and applying extremal length. (See  for the basic properties of extremal length.)

We now show that $`\overline{E}_m<0`$ for $`\mathrm{\Gamma }`$. Note that the excess is positive only on vertices on the boundary of $`2`$-gons, which arise from leaves in $`T`$. On the other hand, every vertex of degree $`3`$ in $`T`$ gives rise to vertices in $`\mathrm{\Gamma }`$ with negative excess. Take as a basepoint for $`\mathrm{\Gamma }`$ a vertex $`w_0S(v_1)`$ with negative excess, say. It is easy to see that there are constants, $`a>1,c>0`$ such that the number $`n_r^{}`$ of negative excess vertices in the combinatorial ball $`B(w_0,r)`$ about $`w_0`$ satisfies $`ca^rn_r^{}a^r/c`$.

If $`w`$ is a vertex with positive excess, then there is a unique vertex $`\sigma (w)`$ with negative excess closest to $`v`$; in fact, if $`wS(v)`$, then $`\sigma (w)`$ is the closest vertex to $`w`$ on $`S(v)`$, and the (combinatorial) distance from $`w`$ to $`\sigma (w)`$ is our parameter $`s`$. The map $`w\sigma (w)`$ is clearly injective. This implies that the number $`n_r^+`$ of positive excess vertices in $`B(w_0,r)`$ satisfies $`n_{r+s}^+n_r^{}`$, $`r\{0,1,2,\mathrm{}\}`$. By choosing $`s`$ sufficiently large, we may therefore arrange to have the total excess in $`B(w_0,r)`$ to be less than $`ϵa^r`$, for some $`ϵ>0`$ and every $`r\{0,1,2,\mathrm{}\}`$. It is clear that the number of vertices with zero excess in $`B(w_0,r)`$ is bounded by a constant (which may depend on $`s`$) times $`n_r^{}`$. Hence, $`\overline{E}_m<0`$ for $`\mathrm{\Gamma }`$.

By allowing $`s`$ to depend on the vertex in $`T`$, if necessary, we may arrange to have $`\underset{¯}{E}_m=\overline{E}_m`$; that is, $`E_m`$ exists, while maintaining $`E_m<0`$. We have thus demonstrated that the resulting surface is a counterexample in $`F_4`$ to the second implication in Nevanlinna’s problem.

## 3. A non-positive curvature example

We now construct an example of a simply connected, complete, parabolic surface $`Y`$ of nowhere positive curvature, with the property

(1)
$$_{D(a,r)}\text{curvature}<ϵarea\left(D(a,r)\right),$$

for some fixed $`aY`$ and every $`r>0`$, where $`D(a,r)`$ denotes the open disc centered at $`a`$ of radius $`r`$, and $`ϵ>0`$ is some fixed constant.

Consider the surface $`=^2`$ with the metric $`|dz|/y`$ in $`P=\{z=x+iy:y1\}`$, and $`\mathrm{exp}(1y)|dz|`$ in $`Q=\{y<1\}`$. We denote this surface by $`Y`$. Let $`\beta `$ denotes the curve $`\{y=1\}`$ in $`Y`$, i.e., the common boundary of $`P`$ and $`Q`$.

Let $`Q^{}`$ denote the universal cover of $`\{z:|z|>1\}`$. Note that $`Q`$ is isometric to $`Q^{}`$ via the map $`z\mathrm{exp}(iz+1)`$. Hence, the curvature is zero on $`Q`$, and the geodesic curvature of $`Q`$ is $`1`$. The geodesic curvature of $`P`$ is $`1`$. Consequently, $`Y`$ has no concentrated curvature on $`\beta `$. The surface $`Y`$ is thus a “surface of bounded curvature”, also known as an Aleksandrov surface (see , ). The curvature measure of $`Y`$ is absolutely continuous with respect to area; the curvature of $`Y`$ is -1 (times area measure) on $`P`$ and $`0`$ on $`Q`$.

The surface $`Y`$ is parabolic, and the uniformizing map is the identity map onto $`^2`$ with the standard metric.

We will now prove (1) with $`a=i`$. Set $`\beta _r=D(a,r)\beta `$. Note that the shortest path in $`Y`$ between any two points on $`\beta `$ is contained in $`P`$, and is the arc of a circle orthogonal to $`\{y=0\}`$. Using the Poincare disc model, it is easy to see that there exists a constant $`c>0`$, such that

(2)
$$ce^{r/2}length\beta _re^{r/2}/c,$$

where the right inequality holds for all $`r`$, and the left for all sufficiently large $`r`$. By considering the intersection of $`D(a,r)`$ with the strip $`1<y<2`$ it is clear that

(3)
$$O(1)area\left(PD(a,r)\right)length\beta _r,$$

for all sufficiently large $`r`$.

Consider some point $`pQ`$, and let $`p^{}`$ be the point on $`\beta `$ closest to $`p`$. It follows easily (for example, by using the isometry of $`Q`$ and $`Q^{}`$) that if $`q`$ is any point in $`\beta `$, then $`d_Q(p,q)=d_Q(p,p^{})+d_Q(p^{},q)+O(1)`$. Consequently, if $`d(p,a)r`$, then there is an $`s[0,r]`$ such that $`p^{}\beta _s`$ and $`d_Q(p,p^{})rs+O(1)`$. Furthermore, it is clear that the set of points $`p`$ in $`Q`$ such that $`p^{}\beta _s`$ and $`d_Q(p,p^{})t`$ has area $`O(t^2+t)length\beta _s`$. Consequently,

$$area\left(QD(a,r)\right)O(1)\underset{j=0}{\overset{r}{}}(j+1)^2length\beta _{rj}.$$

Using (2), we have

(4)
$$area\left(QD(a,r)\right)O(1)length\beta _r,$$

for all sufficiently large $`r`$.

Now, combining (3) and (4), we obtain (1) for all sufficiently large $`r`$. It therefore holds for all $`r`$.

Acknowledgements. The authors are grateful to B. Davis, D. Drasin, and especially to A. Eremenko for helpful discussions and their interest in this work.


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# A quantitative model for presynaptic free Ca^limit-from2+ dynamics during different stimulation protocols

## 1 Introduction

$`\mathrm{Ca}^{2+}`$ is an important cellular messenger. In particular, $`\mathrm{Ca}^{2+}`$ plays a key role in synaptic transmission. For example, it triggers the secretion of neurotransmitter  and is involved in synaptic plasticity  being most likely related to the neural bases of memory . Important phenomena in this context are presynaptic facilitation, short-term potentiation (STP), long-term potentiation (LTP) and long-term depression (LTD). It has been shown that the induction of long-term effects (LTE) are related to a prior $`\mathrm{Ca}^{2+}`$ influx into the neuron .

There exist several theoretical and numerical approaches to describe intracellular calcium dynamics. Some models investigate presynaptic $`\mathrm{Ca}^{2+}`$ dynamics in response to single action potentials . The role of free calcium in transmitter release and in synaptic facilitation has been discussed in detail . These models focus on the geometry of the presynaptic compartment and the distribution of calcium transporting proteins in the membrane. The role of intracellular organelles with the capability to store and release free calcium (e.g. endoplasmatic and sarcoplasmatic reticula) has been analyzed . Also the role of slow and fast intracellular buffers has been investigated . Indeed, most of the incoming calcium ions are bound to some intracellular buffers , such that this issue is very important.

The philosophy of the above models is to focus on the spatial distribution of calcium ions in different compartments of the cell. The connection of calcium and transmitter release has been investigated mostly in two giant synapses: the calyx of Held  and the squid giant synapse . For example it has been discussed for the squid giant synapse that the calcium enhancement in the direct neighborhood of the channels is related to transmitter release and facilitation . To this end the channels enter this model as discrete entities. We would like to reconsider this approach on the basis of new data that are available today.

Single channel properties have become more and more available . In the time of the work of Fogelson and Zucker the exact single protein currents and open dynamics were not available. Therefore, these data were modeled with some free parameters. The question we would like to address is whether we have sufficient information on single protein properties at hand in order to explicitly and quantitatively describe the activation and activity of each protein involved in the transmembrane calcium flux.

To this end we will proceed as follows:

* Instead of describing large squid giant synapse we restrict ourselves in a first step to rather small synapses. We focus on the average calcium transient in the synapse and neglect the effect of calcium diffusion. This prevents us from considering LTE or synaptic facilitation in detail due to the lack of detailed spatial information on inhomogeneous calcium distributions. The average calcium transient is nevertheless an important indicator since the exact amplitude and time course of calcium, i.e. the exact shape of the calcium transient, is believed to be important for transmitter release . The average calcium transient is also suitable for the analysis of the quality of a model based on single protein properties.
* The calcium dynamics is described by well known ordinary differential equations. Within these equations the single protein properties are isolated and treated separately.
* The model parameters concerning the behaviour of single proteins are determined on the basis of independent single protein experiments. This set of parameters will be called universal because it will be assumed that the properties of the proteins do not primarily depend on the neuron under consideration. This is surely an approximation which has to be further validated.
* The model parameters concerning the specific setup of an experiment, i.e. extension of the synaptic compartment, calcium concentrations, stimulation protocol, use of fluorescence indicator, etc. are adjusted to a specific experiment measuring calcium transients in response to various stimulation protocols in single boutons of pyramidal neurons of the rat neocortex . These specific parameters have to be adjusted for each experiment separately.
* The remaining parameters (in our case this will be the protein densities in the membrane of the neuron) are used in order to fit the data. It is important that the number of fit parameters is minimized in order to get a suitable test of the model.

Note, that the separation of universal and specific parameters in the model opens the possibility to use the universal parameters, which have been determined once in independent experiments, for different experimental setups. On one hand, this considerably reduces the number of fit parameters and strongly restricts possible outcomes of the model. On the other hand, this allows to compare the results of different experiments using different protocols or calcium indicators. Indeed, it has been mentioned before, that the amount and type of calcium indicator changes the calcium transients in a non-negligible way . Therefore, we consider this model approach as a first step towards a link between different experiments opening the possibility of a comparative approach to calcium data.

Our main interest is a quantitative description of presynaptic free $`\mathrm{Ca}^{2+}`$ dynamics in response to single action potentials and to different stimulation protocols. We apply the model to single boutons of the pyramidal neurons in the rat neocortex and compare the results to the corresponding experiment . This experiment provides most of the necessary informations in order to determine the specific parameters of the model. We will look for characteristics in $`\mathrm{Ca}^{2+}`$ time courses which are specific for the used stimulation protocols. Furthermore, we will analyze the influence of the calcium indicator on the results of the experiment and discuss the possibility to reconstruct the calcium transient as it would have been without indicator.

## 2 Methods

The intracellular free $`\mathrm{Ca}^{2+}`$ dynamics due to a predefined, time dependent membrane potential of a presynaptic neural domain is described by using a set of ordinary differential equations. This neural domain is represented by a cell membrane which separates the extracellular space from the intracellular space. Specific membrane proteins are embedded into this membrane. Some of these membrane proteins and in addition some intracellular proteins determine the dynamics of the free $`\mathrm{Ca}^{2+}`$ ions concentration .

The model geometry consists of a homogenous reaction volume (one-compartment) which is situated in a homogeneous extracellular space. We are using a spherical symmetry of the reaction volume. However, the results remain valid for other shapes provided that the surface to volume ratio is conserved. In general presynaptic boutons are relatively small, i.e. having a diameter of the order of microns . In this study we assume that the process of diffusion of $`\mathrm{Ca}^{2+}`$ ions is fast enough to reach an equilibrium distribution on a time scale of one $`\mathrm{ms}`$. This assumption has to be reviewed considering larger reaction volumes as found in postsynapses. Consequently, also the surface densities of membrane proteins have to be interpreted as average values over the whole reaction surface.

Starting from a cell in equilibrium the application of a depolarizing membrane potential $`V`$ induces a free $`\mathrm{Ca}^{2+}`$ ion current between the reaction volume and the extracellular space through diverse membrane proteins. This leads to a change of the intracellular free $`\mathrm{Ca}^{2+}`$ concentration $`c=\left[\mathrm{Ca}_{\mathrm{free}}^{2+}\right]`$ which is represented by an ordinary differential equation

$$\frac{d}{dt}c=\frac{G}{\mathrm{zF}}\left\{J_\mathrm{i}(V\left(t\right),\overline{V}\left(c\right))J_\mathrm{e}\left(c\right)+L\right\}+B_{\mathrm{en}}\left(c\right)+B_{\mathrm{ex}}\left(c\right)$$
(1)
in analogy to other studies (see for example  eq.(1) or  eq.(9)). Every source and every sink of the free $`\mathrm{Ca}^{2+}`$ ions is determined by its own term. $`J_\mathrm{i}`$ is the influx current density per membrane surface unit through voltage-dependent $`\mathrm{Ca}^{2+}`$ channels (VDCC) . It directly depends on the applied membrane potential $`V`$ (the time dependence in Eq. (1) is noted explicitly to emphasize that $`V\left(t\right)`$ is treated as input signal), and the effective reversal potential $`\overline{V}`$ and is described in detail in section 2.1. $`J_\mathrm{e}`$ is the efflux current density per membrane surface unit caused by ATP-driven plasma membrane $`\mathrm{Ca}^{2+}`$ pumps (PMCA)  and $`\mathrm{Na}^+`$/$`\mathrm{Ca}^{2+}`$ exchangers (NCX) . It depends on the free intracellular $`\mathrm{Ca}^{2+}`$ concentration $`c`$ and is described in detail in section 2.3. The leakage surface current density $`L`$ is determined by the steady state conditions and represents all currents which are not described in our model in an explicit manner and that determine the equilibrium state (section 2.6). The terms $`B_{\mathrm{en}}`$ and $`B_{\mathrm{ex}}`$ stand for the action of endogenous and the exogenous buffers , respectively, which bind to and disengage from the free $`\mathrm{Ca}^{2+}`$ ions (see section 2.2). The geometry factor $`G=O/V`$ is the surface to volume ratio. It translates the current densities ($`J_\mathrm{i}`$, $`J_\mathrm{e}`$, $`L`$ ) into changes of free intracellular $`\mathrm{Ca}^{2+}`$ concentration. $`\mathrm{z}`$ and $`\mathrm{F}`$ are the valence charge of $`\mathrm{Ca}^{2+}`$ ions and the Faraday constant, respectively.

Note, that the separation of universal and specific parameters is not reflected in Eq. (1). However, in the following sections the explicit form of the currents entering the equation will be developed on the level of single membrane proteins. It is at this point that the parameter classification will be mirrored in the mathematical prescription.

### 2.1 $`\mathrm{Ca}^{2+}`$ influx through VDCC

The VDCC is characterized by a current-voltage relation based on both a voltage dependent current through the open pore $`I_{\mathrm{open}}^{\mathrm{single}\mathrm{channel}}`$ as well as a voltage dependent opening probability $`g_\mathrm{v}`$ . We use the following description of the $`\mathrm{Ca}^{2+}`$ influx current density $`J_\mathrm{i}`$ through VDCC

$$\begin{array}{cc}J_\mathrm{i}(V\left(t\right),\overline{V}\left(c\right))=& \rho _\mathrm{v}g_\mathrm{v}\left(V\left(t\right)\right)\underset{}{\overline{g}_\mathrm{v}\left(\overline{V}\left(c\right)V\left(t\right)\right)}.\\ & I_{\mathrm{open}}^{\mathrm{single}\mathrm{channel}}\end{array}$$
(2)
$`J_\mathrm{i}`$ is weighted with the surface density of the channels $`\rho _\mathrm{v}`$ being the most important cell-type *specific* parameter. Practically all other parameters defining the single channel properties belong to the class of universal parameters. The $`\mathrm{Ca}^{2+}`$ current through the open pore $`I_{\mathrm{open}}^{\mathrm{single}\mathrm{channel}}`$ is driven by the electrochemical gradient over the membrane . A current caused by changes of the membrane potential is followed by changes of the intracellular $`\mathrm{Ca}^{2+}`$ concentration and these retroact on the current. The $`\mathrm{Ca}^{2+}`$ current due to the membrane potential gradient is approximated by a linear voltage-current relation with open pore conductivity $`\overline{g}_\mathrm{v}`$. This is justified for physiologically relevant membrane potentials for which the VDCC current-voltage relation has been found to be indeed nearly linear . The important potential difference entering Eq. (2) is the one relative to the $`\mathrm{Ca}^{2+}`$ reversal potential $`\overline{V}`$ which dynamically incorporates the $`\mathrm{Ca}^{2+}`$ concentration gradient over the membrane into the model using Nernst equation

$$\overline{V}\left(c\right)=\frac{\mathrm{RT}}{\mathrm{zF}}\mathrm{ln}\left(\frac{c_{\mathrm{ext}}}{c}\right)\mathrm{\Delta }V_{\mathrm{eff}}.$$
(3)
$`\mathrm{R}`$ is the molar gas constant and $`\mathrm{T}`$ is the absolute temperature. $`c_{\mathrm{ext}}`$ stands for the external $`\mathrm{Ca}^{2+}`$ concentration. $`\mathrm{\Delta }V_{\mathrm{eff}}`$ corrects the exact reversal potential for the linear approximation. As it has been found that efflux $`\mathrm{Ca}^{2+}`$ currents through VDCC at voltages above the reversal potential are negligible  we set $`I_{\mathrm{open}}^{\mathrm{single}\mathrm{channel}}=0`$ for $`V>\overline{V}`$. Independently from the definition of the reversal potential by the Nernst equation it should be mentioned that its $`\mathrm{Ca}^{2+}`$ dependency has only minor effects on the $`\mathrm{Ca}^{2+}`$ dynamics. Note that the exact value of $`\mathrm{\Delta }V_{\mathrm{eff}}`$ may depend on the ion concentrations in each cell.

The time dependence of the single channel open probability $`g_\mathrm{v}`$ is modeled by a single exponential approximation

$$\frac{d}{dt}g_\mathrm{v}=\left(\widehat{g}_\mathrm{v}\left(V\right)g_\mathrm{v}\right)\frac{1}{\tau }$$
(4)
which may be related to experiment using data for the average number of open channels in an ensemble of channels . The open probability approaches its asymptotic value $`\widehat{g}_\mathrm{v}(V)`$ with a time constant $`\tau `$. $`\widehat{g}_\mathrm{v}(V)`$ is different for each membrane potential and is described by a sigmoidal function

$$\widehat{g}_\mathrm{v}\left(V\right)=\frac{1}{\mathrm{exp}\left(\left(V_\mathrm{h}V\right)\frac{1}{\kappa }\right)+1}$$
(5)
being the best approximation to experimental data . Here, $`V_\mathrm{h}`$ is the half activation voltage and $`\kappa `$ describes the steepness of the asymptotic opening probability $`\widehat{g}_\mathrm{v}`$.

### 2.2 $`\mathrm{Ca}^{2+}`$ buffering via endogenous and exogenous buffers

The main part of the intracellular $`\mathrm{Ca}^{2+}`$ is bound to an endogenous buffer . In addition, also a fluorescence indicator used in experiments acts as an exogenous buffer . In the rest state of the neuron the amount of free buffers is still large enough such that during stimulation the dominant part of incoming free $`\mathrm{Ca}^{2+}`$ ions bind to these buffers . The steadily ongoing binding and dissociation process of $`\mathrm{Ca}^{2+}`$ and buffers strongly influence the resulting free $`\mathrm{Ca}^{2+}`$ dynamics. This applies not only to the phase of growing $`\mathrm{Ca}^{2+}`$ concentration during stimulation but also to the return of the $`\mathrm{Ca}^{2+}`$ concentration to the base level. Therefore, we include both types of buffers explicitly in the model using the following kinetic equations

$$\frac{d}{dt}b_{\mathrm{en},\mathrm{ex}}=k_{\mathrm{en},\mathrm{ex}}^{}b_{\mathrm{en},\mathrm{ex}}+k_{\mathrm{en},\mathrm{ex}}^+c\left(b_{\mathrm{en},\mathrm{ex}}^0b_{\mathrm{en},\mathrm{ex}}\right)$$
(6)
for the endogenous and exogenous buffer . Here, $`b_{\mathrm{en}}`$ and $`b_{\mathrm{ex}}`$ are the concentrations of bound buffers, $`b_{\mathrm{en}}^0`$ and $`n_{\mathrm{ex}}^0`$ are the total concentrations of the intracellular buffer proteins. $`k_{\mathrm{en},\mathrm{ex}}^+`$ and $`k_{\mathrm{en},\mathrm{ex}}^{}`$ are the rate constants in units of $`\frac{1}{\mu \mathrm{M}\mathrm{ms}}`$ and $`\frac{1}{\mathrm{ms}}`$, respectively. As we do not take diffusion into account, we neglect all spatial effects of mobile buffers. Also all effects related to different types of buffers are neglected. Note, that the source and the sink terms for the buffers (Eq. (6)) are the sink and the source terms, respectively, for the $`\mathrm{Ca}^{2+}`$ dynamics (Eq. (1))

$$B_{\mathrm{en},\mathrm{ex}}\left(c\right)=\frac{d}{dt}b_{\mathrm{en},\mathrm{ex}}.$$
(7)
The rate constants are in general not available from experiment. We use the realistic assumption that the buffer dynamics takes places on short time scales compared to the $`\mathrm{Ca}^{2+}`$ dynamics, i.e. that the buffer dynamics is always in a quasi steady state (rapid buffer approximation ). Note, that we neglect low affinity endogenous buffers in the present model. The rapid buffer approximation has two important consequences. First, on the short time scale the bound endogenous and exogenous buffer concentrations $`b_{\mathrm{en},\mathrm{ex}}`$ become constant and can be written as

$$b_{\mathrm{en},\mathrm{ex}}\left(c\right)=\frac{cb_{\mathrm{en},\mathrm{ex}}^0}{K_{\mathrm{en},\mathrm{ex}}+c}.$$
(8)
This implies that the bound buffer concentration is adapted instantaneously to the free $`\mathrm{Ca}^{2+}`$ concentration at each time point $`t.`$ Therefore, we have

$$B_{\mathrm{en},\mathrm{ex}}^{\mathrm{qss}}\left(c\right)=\frac{d}{dt}b_{\mathrm{en},\mathrm{ex}}\left(c\right)=\frac{d}{dc}b_{\mathrm{en},\mathrm{ex}}\left(c\right)\frac{d}{dt}c.$$
(9)
With the help of Eq. (8) and Eq. (9) the $`\mathrm{Ca}^{2+}`$ dynamics (Eq. (1)) gets a more compact form:

$$\frac{d}{dt}c=\frac{G}{\mathrm{zF}}\left\{J_\mathrm{i}J_\mathrm{e}+L\right\}\frac{1}{1+T_{\mathrm{en}}+T_{\mathrm{ex}}}.$$
(10)
$`T_{\mathrm{en}}`$ and $`T_{\mathrm{ex}}`$ are dimensionless variables:

$`T_{\mathrm{en}}\left(c\right)`$ $`=`$ $`{\displaystyle \frac{b_{\mathrm{en}}^0K_{\mathrm{en}}}{\left(K_{\mathrm{en}}+c\right)^2}}\mathrm{with}\mathrm{K}_{\mathrm{en}}={\displaystyle \frac{\mathrm{k}_{\mathrm{en}}^{}}{\mathrm{k}_{\mathrm{en}}^+}}`$
$`T_{\mathrm{ex}}\left(c\right)`$ $`=`$ $`{\displaystyle \frac{b_{\mathrm{ex}}^0K_{\mathrm{ex}}}{\left(K_{\mathrm{ex}}+c\right)^2}}\mathrm{with}\mathrm{K}_{\mathrm{ex}}={\displaystyle \frac{\mathrm{k}_{\mathrm{ex}}^{}}{\mathrm{k}_{\mathrm{ex}}^+}}`$ (11)
and $`\frac{1}{1+T_{\mathrm{en}}+T_{\mathrm{ex}}}`$ is a correction factor in Eq. (10) which depends only on the free $`\mathrm{Ca}^{2+}`$ concentration. The great technical advantage of this approximation  is that Eq. (10) only involves the dissociation constants $`K_{\mathrm{en},\mathrm{ex}}`$ of the buffers instead of both rate constants $`k_{\mathrm{en},\mathrm{ex}}^+`$ and $`k_{\mathrm{en},\mathrm{ex}}^{}`$. In general the dissociation constants are available from experiment.

### 2.3 $`\mathrm{Ca}^{2+}`$ efflux through PMCA and NCX

The PMCA and the NCX are proteins which actively transport $`\mathrm{Ca}^{2+}`$ ions through the membrane  and therefore have to be described differently from pores. The PMCA pumps $`\mathrm{Ca}^{2+}`$ ions against the electrochemical gradient using the energy of ATP molecules. The NCX transports $`\mathrm{Ca}^{2+}`$ ions out of the neuron by exchanging them with $`\mathrm{Na}^+`$ ions. The $`\mathrm{Ca}^{2+}`$ dependence of the PMCA-kinetics observed in experiments is well described by the Hill equation . The activity of the PMCA and the NCX is limited to a maximum rate  which follows from the molecular structure of the proteins. Correspondingly, the free $`\mathrm{Ca}^{2+}`$ efflux in Eq. (1) gets the form

$$J_\mathrm{e}\left(c\right)=\rho _\mathrm{p}g_\mathrm{p}\left(c\right)\overline{I}_\mathrm{p}+\rho _\mathrm{x}g_\mathrm{x}\left(c\right)\overline{I}_\mathrm{x}.$$
(12)
This is again formulated on the single protein level, where $`\rho _{\mathrm{p},\mathrm{x}}`$ represent the *specific* surface densities of the membrane proteins and $`\overline{I}_{\mathrm{p},\mathrm{x}}`$ the *universal* maximum activity rates of the PMCA and the NCX, respectively. For the *universal* activity characteristics $`g_{\mathrm{p},\mathrm{x}}`$ we assume Hill equations

$$g_\mathrm{p}\left(c\right)=\frac{\left(c\right)^{n_\mathrm{p}}}{\left(c\right)^{n_\mathrm{p}}+\left(H_\mathrm{p}\right)^{n_\mathrm{p}}}\text{and}g_\mathrm{x}\left(c\right)=\frac{\left(c\right)^{n_\mathrm{x}}}{\left(c\right)^{n_\mathrm{x}}+\left(H_\mathrm{x}\right)^{n_\mathrm{x}}}$$
(13)
which directly depend on the free $`\mathrm{Ca}^{2+}`$ concentration $`c`$ . $`H_{\mathrm{p},\mathrm{x}}`$ are the half activation concentrations and $`n_{\mathrm{p},\mathrm{x}}`$ the Hill coefficients. This is justified in the case of PMCA  while no explicit measurements exist in the case of NCX. Nevertheless, it is a reasonable assumption that the principal behavior is also well described by a Hill equation.

### 2.4 Universal properties of the model

The classification of the model parameters represents an important element of our model because it enables us to separate those properties which are basically independent of the cell-type from those which are sensitive for differences between cell-types, compartments, and experimental setups. The universal properties of the proteins are postulated to remain invariant for all kinds of neurons and we extracted these values from various single-protein experimental results. The values of universal parameters for VDCC, PMCA and NCX as used in the model are summarized in Tab. 1.

VDCC. These channel-proteins are multi-subunit complexes which form a voltage sensitive transmembrane pore. Six types of VDCC are known, corresponding to the pharmacological properties (i.e. L-, N-, P/Q-, R-type as high-voltage-activated (HVA) channels and T-type as low-voltage-activated (LVA) channels. These types are primarily characterized by genes encoding a different subunit (e.g. the $`\alpha _1`$ subunit) but an overall matching percentage of nearly $`50\%`$ (for HVA channels only) . The $`\mathrm{Ca}^{2+}`$ influx into the presynaptic terminal is dominated by the P/Q- and N-type channels . Each type of VDCC should have the same biophysical properties in all kinds of tissues and animals. Unfortunately the actual precision of the experiments, especially taking into account the variety of experimental setups, does not allow to quantitatively identify the biophysical properties corresponding to each channel type . The resulting uncertainties dominate the differences between the channel types. Therefore, we chose the values from one single channel measurement of N-type channels considering it as a representative member for the VDCC. However, if more conclusive values for different VDCC-types are found in future we may disentangle the influence of different sub-types on $`\mathrm{Ca}^{2+}`$ transients. Concerning the measurements of the P/Q- and N-type channels two additional problems exist. Most measurements were performed with $`\mathrm{Ba}^{2+}`$ as carrier ion and the obtained conductivity differs from the $`\mathrm{Ca}^{2+}`$ conductivity . Additionally all measurements were made at room temperature ($`2025^{}\mathrm{C}`$) and the biophysical properties are temperature dependent . Thus, the obtained $`\mathrm{Ca}^{2+}`$ influx through the VDCC differs between measurements made at room temperature from those which were made at blood temperature. We neglect both deviations because we could neither find convincing extrapolations of the temperature behaviour nor a sufficient relation of different charge carrier ions. Anyhow, those deviations stay within the current experimental accuracy.

PMCA. There exist four different genes which encode the PMCA (PMCA1-4) and all of them occur in different splicing variants (labeled by small letters). These iso-forms have different kinetic properties but again we assume that one iso-form should have the same properties in each type of tissues and animal. The dominant iso-forms of PMCA in the rat brain are the 1a-, 2a-, 2b-, 3a-, 3b-, and 4b-type . Similarly to the VDCC the measured kinetic properties vary considerably due to the different experimental conditions for one type of PMCA  and we chose as representative member the PMCA2a protein. The Hill coefficient and the concentration of half activation are taken from a measurement for rat PMCA  and the maximum pump current is calculated from . We neglect other regulation mechanisms of the PMCA-activity e.g. the roles of calmodulin or of ATP (which is considered to be available to a sufficient degree).

NCX. In the case of the NCX three different genes (NCX1-3) with different splicing variants are known. Here, we do not specify the type of NCX. We chose average values for the maximum current  and for the concentration of half activation , and set the Hill coefficient of the NCX to one. It is worth emphasizing that our results are not significantly altered for other Hill coefficient. Other regulation mechanisms (e.g. the dependence on $`\mathrm{Na}^+`$) are not considered in the model.

### 2.5 Specific properties of the model

Per definition the specific parameters have to be adjusted to the specific neuron type and compartment geometry in the experiment under consideration. Especially, this concerns the rest state properties (i.e. geometry factor $`G`$, rest state membrane potential $`V_0`$, rest state intracellular $`c_0`$ and extracellular $`c_{\mathrm{ext}}`$ $`\mathrm{Ca}^{2+}`$ concentration) which in general are indicated in the experiments. This applies not necessarily to the dissociation constant $`K_\mathrm{m}`$ and the total concentration $`m_0`$ of the endogenous buffer. Here we chose Calmodulin as representative member of buffer molecules and we assume that the four buffer binding sites are independent. Our aim is to quantitatively compare the results of our model with experiments. Because all visualizations of $`\mathrm{Ca}^{2+}`$ dynamics in neural compartments are performed with fluorescence indicators we have to include the parameters specific for the used type of indicator. The corresponding dissociation constant can be extracted from corresponding data sheets and the indicator concentration is usually indicated in the experimental protocol.

The most critical specific parameters are the membrane protein surface densities (e.g. VDCC $`\rho _\mathrm{v}`$, PMCA $`\rho _\mathrm{p}`$). They are rarely known for specific cell types. Even more seldom they are indicated in specific experiments. In addition measurements of protein densities are usually valid for very specific areas on the cell membrane. It is difficult to extrapolate those densities to an average density on a whole presynapse as it is used in the model. As our main interest points towards the analysis of $`\mathrm{Ca}^{2+}`$ transients in response to various stimulation protocols we proceed as follows. We use single action potential $`\mathrm{Ca}^{2+}`$ transients measured as fluorescence signal to fit the unknown protein densities. Then the values found are retained and used for the analysis of $`\mathrm{Ca}^{2+}`$ transients after other stimulation protocols. This procedure is explained in more details in section 3.1.

### 2.6 Equilibrium, stability and stimulation

To obtain a realistic simulation of $`\mathrm{Ca}^{2+}`$ dynamics the model has to recover a stable equilibrium state after some stimulation. That is achieved by the requirement that in the rest state of the neuron (defined by the rest membrane potential $`V_0`$ and the free rest $`\mathrm{Ca}^{2+}`$ concentration $`c_0`$) the leak conductivity of the membrane $`L`$ exactly compensates the netto current densities coming from VDCC ($`J_\mathrm{i}`$), PMCA and NCX ($`J_\mathrm{e}`$).

After application of any type of stimulation the free $`\mathrm{Ca}^{2+}`$ concentration indeed returns to the rest concentration. We did not find any instabilities of the $`\mathrm{Ca}^{2+}`$ dynamics within physiologically relevant stimulation protocols. The model results are robust against variation of any parameters on the qualitative level. Quantitatively the results are most sensitive to variations of the membrane protein surface densities and of the dissociation constant of the endogenous buffer.

For the stimulation protocols (single action potential and trains of action potentials) we use membrane potentials $`V\left(t\right)`$ which are simulated with a system of coupled differential equations (Hodgkin-Huxley like, not shown here). The parameters of those equations have to be adjusted such that the stimulation shapes as found in the experiment under consideration are appropriately reproduced.

## 3 Results

To check if our model correctly describes the presynaptic $`\mathrm{Ca}^{2+}`$ dynamics in response to single action potentials, we tuned the model with respect to an experiment measuring the $`\mathrm{Ca}^{2+}`$ dynamics in presynaptic boutons of pyramidal neurons in the neocortex of rats . The corresponding $`\mathrm{Ca}^{2+}`$ transients can be reproduced for reasonable membrane protein surface densities. Using those parameters we can calculate the $`\mathrm{Ca}^{2+}`$ dynamics due to a $`10\mathrm{Hz}`$ tetanus and compare the result to corresponding measurements  (see section 3.1). A qualitative discussion of $`\mathrm{Ca}^{2+}`$ transients in response to various stimulation protocols is provided in section 3.2. Finally we will quantitatively illustrate the influence of fluorescence indicators on $`\mathrm{Ca}^{2+}`$ dynamics and discuss the implications for experiments using fluorescence indicators to measure $`\mathrm{Ca}^{2+}`$ transients (see section 3.3).

### 3.1 Quantitative model test

At first we verify that our model can describe $`\mathrm{Ca}^{2+}`$ transients evoked by single action potentials in single presynaptic boutons from pyramidal neurons in the neocortex of rats on the basis of a very detailed corresponding fluorescence measurements . The universal parameters should especially apply for this system and are used as shown in Tab. 1. The specific parameters are determined directly and indirectly by the measurement  and by using independent experimental results. We calculated the geometry factor with the help of a fluorescence figure of the whole bouton (Fig. 3A in ). We determined the dissociation constant of the endogenous buffer $`K_\mathrm{m}`$ using the observation that approximately only $`1\%`$ of the total incoming intracellular $`\mathrm{Ca}^{2+}`$ remains free . The surface density of the NCX $`\rho _\mathrm{x}`$ was determined with the help of a measurement which investigates its ratio to the PMCA density $`\rho _\mathrm{p}`$ . The values for the specific parameters are summarized in Tab. 2. They have to be carefully interpreted as an approximation for the neuron type under consideration. Please note that only two parameters remained free to adjust the model to the experimental data: The densities of the PMCA and of the VDCC. Note, that any dependence of channel densities on intracellular $`\mathrm{Ca}^{2+}`$  has not been considered here.

In general $`\mathrm{Ca}^{2+}`$ measurements in neurons use fluorescence indicators to visualize the $`\mathrm{Ca}^{2+}`$ concentration and distribution. As already pointed out such fluorescence indicators act as an exogenous buffer system which, therefore, has been included into the model prescription (see section 2.2). With the help of a $`1:1`$ complexation ansatz  we translated the simulation of the $`\mathrm{Ca}^{2+}`$ dynamics into a simulation of the fluorescence signal

$$\mathrm{\Delta }f/f=\left(\mathrm{\Delta }f/f\right)_{\mathrm{max}}\frac{c\left(t\right)c_0}{c\left(t\right)+K_{\mathrm{ex}}}.$$
(14)
$`\mathrm{\Delta }f/f`$ is the relative fluorescence change, $`\left(\mathrm{\Delta }f/f\right)_{\mathrm{max}}`$ represents its maximum, and $`K_{\mathrm{ex}}`$ is the dissociation constant of the indicator. The properties of the used indicators (Magnesium Green (Molecular Probes)) are shown in Tab. 2. Note, that in the experiment different concentrations of indicator have been used .

The only remaining free parameters are the surface densities of the VDCC $`\rho _\mathrm{v}`$ and the PMCA $`\rho _\mathrm{p}`$. We simulate the amplitude and the shape of the action potential as applied to the presynapse in the experiment and fit the protein densities (see Tab. 2) such that the simulation result agrees with the measured fluorescence signal evoked by a single action potential (Fig. 15C in ). Note that the experiment has been executed with $`100\mu \mathrm{M}`$ Magnesium Green and $`\left(\mathrm{\Delta }f/f\right)_{\mathrm{max}}^{100\mu \mathrm{M}}`$ has to be chosen correspondingly (see Tab. 2). The result is shown in Fig. 1. The amplitude and the shape of the simulated fluorescence response are in good agreement with the data observed in experiment.

The next step is to investigate whether the same model without changing the model assumptions is able to describe $`\mathrm{Ca}^{2+}`$ transients evoked by more complex stimulation protocols. We compare the fluorescence response evoked by a $`10\mathrm{Hz}`$ tetanus to the model predictions. To this end the action potentials in the model have to be adjusted to those used in experiment (Fig. 9; upper panel in ). The experiment has been performed with $`500\mu \mathrm{M}`$ Magnesium Green and we corrected the indicator concentration correspondingly. The maximal relative fluorescence change $`\left(\mathrm{\Delta }f/f\right)_{\mathrm{max}}^{500\mu \mathrm{M}}`$ has not been stated explicitly. Therefore, we determine this value by the requirement that the $`\mathrm{Ca}^{2+}`$ signal in response to a single action potential (Fig. 15C; middle column in ) is reproduced correctly (data not shown). Finally, the protein surface densities are adjusted to reproduce the $`\mathrm{Ca}^{2+}`$ response to the first action potential of the tetanus (see Tab. 2). The subsequent $`\mathrm{Ca}^{2+}`$ signal as predicted by the model turns out to be in perfect agreement with the fluorescence signal (see Fig. 2). Note, that all others parameters of the model remained unchanged. We conclude that the model once fitted to the $`\mathrm{Ca}^{2+}`$ signal in response to single action potentials in a specific type of neuron describes the $`\mathrm{Ca}^{2+}`$ signal due to tetanus stimulation without further changes.

### 3.2 $`\mathrm{Ca}^{2+}`$ dynamics in response to different stimulation protocols

The main question we address is whether there are characteristic differences between the different $`\mathrm{Ca}^{2+}`$ transients or not. In the low frequency domain ($`2\mathrm{Hz},3.5\mathrm{s}`$) the $`\mathrm{Ca}^{2+}`$ response appears as a train of independent single action potential responses (Fig. 3). The $`\mathrm{Ca}^{2+}`$ concentration follows the activation by each action potential. Shape and amplitude of each $`\mathrm{Ca}^{2+}`$ response is unaltered compared to the response to a single action potential. This frequencies range is typically used for LTD induction .

For higher frequencies ($`20\mathrm{Hz},350\mathrm{ms}`$) a new baseline in the $`\mathrm{Ca}^{2+}`$ concentration arises during stimulation (Fig. 4). The $`\mathrm{Ca}^{2+}`$ pumps and the $`\mathrm{Na}^+`$/$`\mathrm{Ca}^{2+}`$ exchangers have not enough time to return the neuron into its rest state. This implies an overlap of $`\mathrm{Ca}^{2+}`$ response to subsequent action potentials. Note, that this qualitative behavior has been observed for example in experiments performed on dendritic spines of pyramidal neurons . However, we can not expect that the presynaptic model also quantitatively describes the $`\mathrm{Ca}^{2+}`$ response in dendritic spines correctly (especially without including diffusion in the model).

This strong increase of the peak and baseline and especially of the average $`\mathrm{Ca}^{2+}`$ concentration for stimulations with higher frequencies is a significant difference compared to the $`\mathrm{Ca}^{2+}`$ response to a low-frequency tetanus. One may suspect that this behaviour is an important part of a process to change synaptic efficiency. For example an accumulation of residual free $`\mathrm{Ca}^{2+}`$ is responsible for synaptic facilitation in presynaptic boutons . Also for the induction of LTP it is well established fact, that the intensity of stimulation protocols have to overcome some threshold . The strong increase of the $`\mathrm{Ca}^{2+}`$ concentration emerges for frequencies that are in the same range as this threshold . This hypothesis also agrees with the experimental fact that a modest $`\mathrm{Ca}^{2+}`$ influx causes LTD, whereas a large $`\mathrm{Ca}^{2+}`$ influx triggers LTP . In general the role of enhanced average $`\mathrm{Ca}^{2+}`$ concentrations for changes of synaptic efficiency has been pointed out by . In order to look for a specific key of a concrete induction process of one of the different forms of synaptic plasticity one has to consider the synapse in more detail. For instance exocytosis steps , buffer properties , diffusion effects  and protein kineses  are important elements of the induction machinery. This is a task for future modeling work.

During the application of a high-frequency tetanus ($`50\mathrm{Hz},1\mathrm{s}`$) the induced $`\mathrm{Ca}^{2+}`$ spikes remain in phase with the stimulating membrane potential. The enhancement of the $`\mathrm{Ca}^{2+}`$ baseline becomes even more pronounced (Fig. 5). Again the new baseline saturates during stimulation but on a higher level compared to the $`20\mathrm{Hz}`$ stimulation. The exact shape of the $`\mathrm{Ca}^{2+}`$ signal is a result of an interplay of membrane proteins and $`\mathrm{Ca}^{2+}`$ buffers. However, we observed some relations between the general appearance of the $`\mathrm{Ca}^{2+}`$ signal and some specific neuron properties. The surface density of VDCC basically determines the amplitude of the $`\mathrm{Ca}^{2+}`$ spike on the top of the baseline. The dissociation constant determines the height of the new baseline. The time scale on which the $`\mathrm{Ca}^{2+}`$ concentration returns to its rest state is governed by the surface densities of the PMCA, NCX and the dissociation constant of the buffer. We emphasize that even higher stimulation frequencies lead to still higher baseline levels of the $`\mathrm{Ca}^{2+}`$ concentration. The reached baseline grows quadratically with the stimulation frequency up to $`100\mathrm{Hz}`$.

### 3.3 Fluorescence indicators disturb the intrinsic $`\mathrm{Ca}^{2+}`$ dynamics

In section 3.2 we have seen that for stimulations with frequencies above some threshold frequency a new baseline of the $`\mathrm{Ca}^{2+}`$ concentration arises. In the following we show that the use of fluorescence indicators for the visualization of $`\mathrm{Ca}^{2+}`$ in experiments alters the $`\mathrm{Ca}^{2+}`$ response and especially the emergence of a new baseline considerably. In Fig. 6 the $`\mathrm{Ca}^{2+}`$ transients evoked by single action potentials with and without fluorescence indicator is compared ($`100\mu \mathrm{M}`$ Magnesium Green). The amplitude of the $`\mathrm{Ca}^{2+}`$ signal is decreased if the fluorescence indicator is used. In addition the $`\mathrm{Ca}^{2+}`$ relaxation time (needed to recover the rest $`\mathrm{Ca}^{2+}`$ concentration) becomes considerably larger using the fluorescence indicator. These relations are not surprising as the indicator binds an important part of incoming free $`\mathrm{Ca}^{2+}`$ ions and reduces the free $`\mathrm{Ca}^{2+}`$ concentration. Note, that the larger relaxation times are not caused by a decreased pump activity which is induced by the lower $`\mathrm{Ca}^{2+}`$ peak. This has consequences for the interpretation of experiments using fluorescence indicators.

The fact that fluorescence indicators act as an additional intracellular buffer changing the shape of the calcium transient has been pointed out before . In Fig. 7 the influence of the fluorescence indicator ($`500\mu \mathrm{M}`$ Magnesium Green) on the $`\mathrm{Ca}^{2+}`$ transients evoked by a $`10\mathrm{Hz}`$ tetanus in a pyramidal neuron of the rat neocortex  is shown. The left panel (with fluorescence indicator) clearly exhibits a new baseline of the $`\mathrm{Ca}^{2+}`$ concentration whereas the baseline remains practically unchanged in the right panel (without fluorescence indicator). Because of the longer relaxation time for the $`\mathrm{Ca}^{2+}`$ spikes the fluorescence indicator facilitates the emergence of a new baseline. Using a fluorescence indicator the $`\mathrm{Ca}^{2+}`$ spikes overlap already for lower frequencies. Consequently, the threshold stimulation frequency for the emergence of a new baseline is higher for neurons without indicator compared to neurons that has been treated with indicator. A interesting conclusion from that fact is that the threshold frequency for the appearance of enhanced $`\mathrm{Ca}^{2+}`$ level changes to lower frequencies if a buffer is present. This may have implications for the induction of LTP in experiments using fluorescence indicators. Note, that the right panel in Fig. 7 provides a quantitative prediction of the $`\mathrm{Ca}^{2+}`$ signal in the pyramidal neuron of the neocortex as it would have been without the use of $`\mathrm{Ca}^{2+}`$ indicator starting from the $`\mathrm{Ca}^{2+}`$ measurement . The same reconstruction procedure may be applied to other experiments in future.

## 4 Discussion

We developed a model for the presynaptic $`\mathrm{Ca}^{2+}`$ dynamics which includes VDCC, PMCA, and NCX, as well as endogenous buffers and fluorescence indicators. Those ingredients turned out to be sufficient to quantitatively reproduce a fluorescence measurement of intracellular free $`\mathrm{Ca}^{2+}`$ transients in response to single action potentials in pyramidal neurons of the rat neocortex. On the basis of the parameter set determined by the single action potential $`\mathrm{Ca}^{2+}`$ transients the model predicts the $`\mathrm{Ca}^{2+}`$ response to $`10\mathrm{Hz}`$ tetanus (using the same neuron type) in a quantitatively accurate way. We conclude that from the point of view of the model $`\mathrm{Ca}^{2+}`$ induced $`\mathrm{Ca}^{2+}`$-release is not necessarily involved into the presynaptic $`\mathrm{Ca}^{2+}`$ dynamics at least for this specific type of neuron.

We would like to emphasize that the single action potential $`\mathrm{Ca}^{2+}`$ response has been produced by fitting only two parameters, i.e. the average membrane protein surface densities of the VDCC and the PMCA. All other parameters have been determined either by the experiment itself or by independent experiments. The resulting values for the VDCC density are in good agreement with experimental measured values . The PMCA density in contrast turn out to be rather high but remain within a range of densities that has been observed in experiment . One may think of inverting the line of argumentation and interprete the resulting protein densities as prediction for an average density on the whole bouton. In view of the difficulties to measure such densities this provides an attractive possibility.

The classification of parameters into universal and specific ones opens the possibility to adjust the model to other experiments without altering the model pillars. The (universal) properties of the VDCC for example have been determined using corresponding single channel experiments. In this context one may think of the necessity to include more than one HVA channel type into the model instead of restricting oneself to one representative type. However, a corresponding analysis revealed that the resulting $`\mathrm{Ca}^{2+}`$ transients remain basically unchanged using two different HVA channels. We conclude that the measurement of $`\mathrm{Ca}^{2+}`$ transients does not allow to distinguish between different HVA channel subtypes. This may be different if considering LVA channels in the model.

We qualitatively investigated the general behaviour of $`\mathrm{Ca}^{2+}`$ transients in response to different stimulation protocols. For $`2\mathrm{Hz}`$ tetanus we found no characteristic feature of the $`\mathrm{Ca}^{2+}`$ signal. This agrees with the interpretation, that synaptic facilitation is triggered by calcium enhancements, while synaptic depression is a consequence of reduced release site activity . We found a characteristic difference between $`\mathrm{Ca}^{2+}`$ transients in response to $`2\mathrm{Hz}`$ and $`50\mathrm{Hz}`$ stimulations. At some threshold stimulation frequency the $`\mathrm{Ca}^{2+}`$ signal does not return to its rest state level after each spike. Instead a new baseline emerges on a higher level and the $`\mathrm{Ca}^{2+}`$ spikes develop on top of this baseline. This general behaviour has also been found in postsynaptic experiments before . Indeed, the strong increase in the $`\mathrm{Ca}^{2+}`$ signal for above threshold stimulation frequencies can be interpreted to correspond to the stimulation threshold for changing synaptic efficiency . In other words, the emergence of an enhanced baseline and, as a consequence, of an enhanced average value and increased peak values of the $`\mathrm{Ca}^{2+}`$ concentration may be thought of as a necessary requirement for induction of changes in the synaptic efficiency. The height of the baseline increases quadratically with increasing stimulation frequencies up to frequencies for which the buffer becomes saturated with $`\mathrm{Ca}^{2+}`$. For higher frequencies the baseline increases only linearly.

Already on the level of calcium transients averaged over the whole bouton we can confirm the result, that the endogenous and exogenous buffer concentrations as well as their saturation properties crucially shift the threshold of the stimulation frequency . Especially, it is important to realize the strong influence of indicators on the intracellular free $`\mathrm{Ca}^{2+}`$ signal . Therefore, we investigated this problem in the framework of the model in more details. On the level of single action potentials the use of indicator leads to considerably smaller $`\mathrm{Ca}^{2+}`$ spikes which relaxed more slowly to the rest concentration. This implies that the use of indicator shifts the above mentioned threshold stimulation frequency. Therefore, a quantitative evaluation of fluorescence measurements should always include the effect of the indicator . The model presented here, provides a tool to reconstruct the averaged $`\mathrm{Ca}^{2+}`$ transient as it would have bean without indicator in fluorescence measurements. This has been done in this article for the experiment of . It would be interesting in future to make use of the parameter classification, to enlarge the model to calcium and buffer diffusion, and to apply the model to other experiments, especially when using other neuron types. A comparative analysis of presynaptic $`\mathrm{Ca}^{2+}`$ transients in different neurons may reveal neuron-type specific characteristics of the $`\mathrm{Ca}^{2+}`$ dynamics.

### Acknowledgments

We are indebted to H. J. Koester and B. Sakmann for providing us with the fluorescence data .


text:
# Errors in Low and Lapsley’s article ”Optimization Flow Control, I: Basic Algorithm and Convergence”

## 1 Error in the proof of the Theorem 1

In the proof of Lemma 3 (at the beginning) on the page 871 it is written: ”Given any $`p,q0`$, using Taylor theorem and Lemma 2 we have $`D(q)D(p)=^2D(w)(qp)=`$ \[…\] for some $`w=tp+(1t)q0,t[0,1]`$.”

The problem is, that the mentioned Taylor theorem, or rather multidimensional mean value theorem, is not true. Here is the counterexample:
Let us denote

$$z=\left[\begin{array}{c}x\\ y\end{array}\right]$$

and take:

$$D(z)=x^2y+y^4$$
(1)
and

$$p=\left[\begin{array}{c}0\\ 0\end{array}\right],q=\left[\begin{array}{c}1\\ 1\end{array}\right]$$

We will get the following gradient of the function $`D(z)`$:

$$D(z)=\left[\begin{array}{c}2xy\\ x^2+4y^3\end{array}\right]$$
(2)
and the following Hessian:

$$^2D(z)=\left[\begin{array}{cc}2y& 2x\\ 2x& 12y^2\end{array}\right]$$
(3)
Then, making use of the mentioned ”theorem”, denoting $`v=1t`$ we will get:

$$\left[\begin{array}{c}2\\ 5\end{array}\right]=\left[\begin{array}{cc}2v& 2v\\ 2v& 12v^2\end{array}\right]\left[\begin{array}{c}1\\ 1\end{array}\right]$$
(4)
Hence, at the same time it must be $`4v=2`$ and $`2v+12v^2=5`$, what is impossible.

## 2 Error in the proof of the Theorem 2

The last passage in the assessment (29) on the page 873 (the proof of Lemma 6) is incorrect. Low and Lapsley justify it: ”where the last inequality holds because the convex function $`_iy_i^2+z^2_iy_iz`$ attains a unique minimum over $`\{(y_i,z)|y_i0,z0\}`$ at the origin.”
But this is not true. It is sufficient to take $`dimy=5`$ and $`y=[5,\mathrm{\hspace{0.17em}4},\mathrm{\hspace{0.17em}3},\mathrm{\hspace{0.17em}4},\mathrm{\hspace{0.17em}5}]`$, $`z=10`$. The considered function takes the value $`19`$.
It is so, because the Hessian of the considered function has the form (taking: $`x=[y_1,y_2,\mathrm{},z]^{}`$):

$$H=\left[\begin{array}{cccccc}2& 0& 0& \mathrm{}& 0& 1\\ 0& 2& 0& \mathrm{}& 0& 1\\ 0& 0& 2& \mathrm{}& 0& 1\\ \mathrm{}& & & & & \\ 0& 0& 0& \mathrm{}& 2& 1\\ 1& 1& 1& \mathrm{}& 1& 2\end{array}\right]$$
(5)
and its characteristic polynomial (e.g. calculated from the Schur’s formula: for $`A:n\times n;`$$`D:m\times m,B^T,C:m\times n;det([A,B;C,D])=det(A)det(DCA^1B)`$ ) will be:

$$det(H\lambda I)=(2\lambda )^{n1}(\lambda ^24\lambda +4n)$$
(6)
where $`n=dimy`$. In this way we will have eigenvalues: $`\lambda _i=2,i=1,\mathrm{},n1`$ and $`\lambda _{n,n+1}=2\pm \sqrt{n}`$. This means, that for $`n>4`$ there will be one negative eigenvalue and the function will not be convex.

## 3 Corrections

The error in the proof of the first theorem was noticed independently by Edward Fan from UCLA in March 2002. The correction is based on the Theorem 9.19 from the Rudin’s book .

The correction of the proof of the second theorem was done by the author and is on the Web page:
http://www.ia.pw.edu.pl/~karbowsk/pub/papers
This paper was submitted to ”IEEE/ACM Transactions on Networking” in August 2002.

According to these suggestions Steven Low corrected his article. The corrected version is now on the Web page:
http://netlab.caltech.edu/pub/papers/ofc1\_ToN.pdf


text:
# Monitoring the Health and Safety of the ACIS Instrument On-Board the Chandra X-ray Observatory

## 1 INTRODUCTION

Just past midnight on July 23, 1999, the space shuttle Columbia lifted-off from Cape Canaveral, Florida. In its payload bay lay the Chandra X-ray Observatory (CXO), the primary cargo of the STS-93 mission. Just under 8 hours after launch, Chandra was deployed from the space shuttle. However, it would be nearly two weeks later, after an Inertial Upper Stage booster “burn” and several “burns” by its own propulsion system, that Chandra would reach its final orbit. The CXO is now the third of NASA’s “great observatories” in space.

The CXO’s operational orbit has an apogee of approximately 140,000 km and a perigee of nearly 10,000 km, with a $`28.5^{}`$ initial inclination. The CXO’s highly elliptical orbit, with an orbital period of approximately 2.65 days, results in high observing efficiency. Moreover, the fraction of the sky occulted by the Earth is small over most of the orbital period, as is the fraction of time when the detector backgrounds are high as the CXO dips into the Earth’s radiation belts. Consequently, approximately 85% of Chandra’s orbit is available for observing. In fact, uninterrupted observations lasting as long as 2.3 days are possible.

The CXO carries two focal plane science instruments: the High Resolution Camera (HRC) and the Advanced CCD Imaging Spectrometer (ACIS). The Observatory also possesses two objective transmission gratings: a Low Energy Transmission Grating (LETG) that is primarily used with the HRC, and the High Energy Transmission Grating (HETG) that is primarily used with the ACIS. In addition to these instruments, Chandra also carries a radiation monitor – the Electron, Proton, Helium Instrument (EPHIN) particle detector.

In order to maintain this high level of observing efficiency, a robust monitoring and alert system must be in place to verify the health and safety status of not only the instruments themselves, but the whole observatory as well. This requirement is a result of the fact that the CXO is not in constant communication with the ground. Communication with the CXO is obtained through NASA’s Deep Space Network (DSN) of ground-tracking stations. Since the DSN supports satellite operations of many different NASA missions, this resource is shared amongst all satellites currently in space.

Chandra’s nominal communication schedule consists of 1-hour contacts spaced approximately 8-hours apart. Therefore, over the course of one 24-hour period, the Chandra X-ray Observatory Center (CXC) receives telemetry from the observatory three times per day for a total of three hours, on average. Of course, during the periods in which the ground is not in contact with Chandra, routine science operations continues autonomously on-board the spacecraft. Each week, a new set of command loads is generated, reviewed, and uplinked to Chandra for use in the following week. These command loads contain all the necessary information for the observatory to execute and perform a week’s worth of science observations and spacecraft maintenance activities. In a companion paper, we review the command load inspection procedure for the ACIS instrument. The science data and observatory health and safety telemetry are stored on Chandra’s solid state recorders (SSR). During a real-time communication contact, these data are telemetered to the ground and transfered to the CXC for data processing (via the Jet Propulsion Laboratory). Another paper in this volume discusses the monitoring and trends analysis of the SSR data. However, since it can take as much as 12-24 hours for the SSR data to be processed, analysed, and hence a putative anomaly found, it is vital that the health and safety status of the Observatory be quickly established during each communication pass. In addition to telemetering SSR data, “live” or real-time telemetry data from the observatory and all instruments, i.e., the current values of all the observatory and instrument house-keeping electronics, are sent immediately to the Chandra Operations and Control Center (OCC) during each DSN communication pass.

In this paper, we focus exclusively on the real-time monitoring of the Advanced CCD Imaging Spectrometer (ACIS) on-board the CXO during these one hour communication contacts. In Section 2, we provide a brief synopsis of the CXO and its primary focal plane instruments while Section 3 illustrates the data flow from spacecraft to monitoring. In Section 4 we discuss the method by which we arrived at our set of health and safety limits. In Section 5, we present the real-time monitoring schemes developed for the ACIS instrument. Our conclusions follow in Section 6.

## 2 CHANDRA X-RAY OBSERVATORY’S FOCAL PLANE INSTRUMENTS

The observatory consists of a spacecraft system and a telescope/science-instrument payload. The spacecraft system provides mechanical controls, thermal control, electric power, communication/command/data management, and pointing and aspect determination. This section briefly describes the two focal plane instruments on-board the CXO, the HRC and the ACIS, while the main emphasis in this paper is the real-time health and safety monitoring of the ACIS instrument. The AXAF Observatory Guide and the AXAF Science Instrument Notebook contain a wealth of information about the CXO and its scientific instruments. More in depth discussions of the Chandra mission, spacecraft, other instruments and subsystems are presented elsewhere. , , , , ,

### 2.1 High Resolution Camera (HRC)

The High Resolution Camera, HRC, is a microchannel plate (MCP) instrument. It is comprised of two detector elements, a $``$ 100 mm square optimized for imaging (HRC-I) and a $``$ 20 x 300 mm rectangular device optimized for the Low Energy Transmission Grating (LETG) Spectrometer readout (HRC-S).

The HRC has the highest spatial resolution imaging on Chandra$``$ 0.5 arcsec (FWHM) – matching the High Resolution Mirror Assembly (HRMA) point spread function most closely. The HRC energy range extends to low energies, where the HRMA effective area is the greatest. The HRC-I has a large field of view (31 arcmin on a side) and is useful for imaging extended objects such as galaxies, supernova remnants, and clusters of galaxies as well as resolving sources in a crowded field. The HRC has good time resolution (16 $`\mu `$sec), valuable for the analysis of bursts, pulsars, and other time-variable phenomena but has limited energy discrimination, E/$`\mathrm{\Delta }`$E $``$ 1 ($`<`$ 1 keV). The HRC-S is used primarily for readout of the low-energy grating, LETG, for which its large format with many pixels gives high spectral resolution ($`>`$ 1000, 40-60 $`\AA `$) and wide spectral coverage (3 - 160 $`\AA `$).

### 2.2 Advanced CCD Imaging Spectrometer (ACIS)

ACIS is the Advanced CCD Imaging Spectrometer. It is comprised of two arrays of CCDs, one optimized for imaging wide fields (ACIS-I; a 2x2 chip array with 16 arcmin on a side), the other optimized for grating spectroscopy and for imaging narrow fields (ACIS-S; a 1x6 chip array; 8 arcmin x 48 arcmin). Each array is shaped to follow the relevant focal surface. In conjunction with the HRMA, the ACIS imaging array provides simultaneous time-resolved imaging and spectroscopy in the energy range $``$ 0.5 - 10.0 keV. When used in conjunction with the High Energy Transmission Gratings (HETG), the ACIS spectroscopic array acquires high resolution (up to E/$`\mathrm{\Delta }`$E = 1000) spectra of point sources. The CCDs had an intrinsic energy resolution (E/$`\mathrm{\Delta }`$E) which varied from $``$5 to $``$50 across the energy range. However, due to radiation damage early in the mission, this has been significantly compromised., Nevertheless, approximately 90% of all Chandra science observation time available during the first four years of the mission has utilized the ACIS instrument.

ACIS employs two varieties of CCD chips. Eight of the 10 chips are “front-side” (or FI) illuminated. That is, the front-side gate structures are facing the incident X-ray beam from the HRMA. Two of the 10 chips (S1 and S3) have had treatments applied to the back-sides of the chips, removing the insensitive, undepleted, bulk silicon material and leaving only the photo-sensitive depletion region exposed. These “back-side”, or BI chips, are deployed with the back side facing the HRMA. BI chips have a substantial improvement in low-energy quantum efficiency as compared to the FI chips because no X-rays are lost to the insensitive gate structures but suffer from poorer charge transfer inefficiency and poorer spectral resolution (prior to launch). In addition, early analysis from on-orbit data indicate that the BI CCDs are more susceptible to “background flares”, which may compromise a measurement, than are FI CCDs. These background flares (i.e., rapid increases in detector background) are thought to be a consequence of Chandra’s radiation environment.

## 3 Outline of Data Flow

As outlined in the Introduction, there are two data streams that are monitored for health and safety issues: (1) data received from the solid state recorder, and (2) “live” or “real-time” data received directly from the CXO via the DSN. In this section, we will briefly outline the data flow associated with the second stream. The former is presented elsewhere in these proceedings.

Once a communication link has been established with a ground-tracking station, real-time telemetry begins flowing immediately to the CXO Operations and Control Center in Cambridge, MA. The relevant ACIS house-keeping telemetry must be extracted from the raw telemetry before any health and safety monitoring may be performed. To accomplish this task, a C++ software package called ACORN (A Comprehensive object-ORiented Necessity) was written by programmers at the CXC.

In our implementation, the real-time raw telemetry stream is fed to ACORN via a User Datagram Protocol (UDP) port. The raw Chandra telemetry consists of approximately 11,000 mnemonic string identifiers (MSIDs). Every spacecraft and instrument sensor, thermistor, meter, etc. are identified and tracked via a unique MSID. ACORN decodes the real-time telemetry stream and writes MSIDs, values, and spacecraft times to either a tab-delimited file or standard output. For our purpose, ACORN-decoded data is written to a tab-delimited file so that it may be analyzed. This entire process of real-time raw spacecraft telemetry to output files that can be processed, is depicted in Figure 1.

## 4 Determining ACIS Health and Safety Limits

In order to monitor the health and safety status of the ACIS instrument, a set of MSIDs as well as a set of limits must first be established. In total, there are 65 ACIS MSIDs that are regularly sent by the instrument to the observatory for incorporation into the telemetry stream that constitute the ACIS hardware “house-keeping telemetry”. These MSIDs consist of components like the camera-body temperature to the ACIS focal plane temperature (please see Figure 2). By and large, these are the principle electronic (i.e., voltages and currents) and temperature MSIDs reported by ACIS.

Prior to launch, as well as during the first 2 years of the mission, there were several sets of “limits” used by various teams within the CXC to ostensibly suit their own particular purpose. The original health and safety limits (i.e., pre-launch) were established based on the qualification tests conducted during the instrumental and observatory thermal-vacuum tests. Therefore, the first task was to update and streamline the use of various limit sets to one definitive set. Two years into the mission, after sufficient experience had been gained with instrument response to on-orbit use, various members of the CXC convened to identify a robust set of limits to check against the real-time ACIS telemetry feed.

It was quickly decided that one set of limits would not be desirable since there are principally two concerns that would govern what the limits should be: (1) health and safety concerns, and (2) data quality concerns. Clearly, the former is more important than the latter since the former is used to monitor and maintain vital mechanical operation whereas the latter serve to indicate possible reduction in data quality. Moreover, the philosophy of the the health and safety limits is such that should a “red” limit violation occur, the CXC response will be immediate. Should a “yellow” limit occur, further analysis may be performed before any response would be initiated. With this operating principle, two sets of limits were created for each MSID, with each set containing caution (color-coded yellow) low and high values and warning (color-coded red) low and high values that would be applicable to health and safety concerns as well as concerns that would affect data quality.

Nearly 300 days worth of data from the second year of the mission were analyzed to arrive at the ACIS health and safety as well as data quality “yellow” and “red” limits. Limits from the MIT/ACIS instrument team were compared against this 300-day baseline and modified on a case-by-case basis. For some MSIDs, the concept of either a low or high “yellow” and/or “red” limit has no meaning and so these MSIDs were assigned a nominal value of “999” to indicate such a condition. These limits are presented in Figure 2. Once this definitive set of limits was determined, they were implemented by every group within the CXC that monitor the telemetry from the spacecraft. One caveat that should be noted is that these MSIDs label telemetered sensor readouts such as the camera-body temperatures and the various voltages and currents generated by the ACIS power supply (see figure 2). The ACIS focal-plane temperature, FP-TEMP, is not included with the MSID values sent to ACORN but is extracted from the Digital Electronics Assembly (DEA) house-keeping packets reported by the ACIS flight software. It is unavailable when the flight software is halted or when the DEA house-keeping is disabled.

## 5 Real-Time Monitoring of the ACIS Instrument

There are two different ways in which the health and safety of the ACIS instrument is monitored. The first is by monitoring and limit-checking the 65 ACIS house-keeping MSIDs discussed in the preceding section. The second method is by monitoring the ACIS science data packets produced by the ACIS flight software system. The former is discussed in Section 5.1 while the latter is discussed in Section 5.2. The principle difference between these two methods is that the former monitors telemetry reported by the hardware components on ACIS, whereas the latter monitors the science telemetry reported by the ACIS flight software.

### 5.1 Monitoring the ACIS House-keeping Telemetry

With the ACIS health and safety limits as well as access to real-time telemetry to perform limit-checking established, we now outline the process by which the ACIS telemetry is monitored and key ACIS personnel notified in case of an anomaly.

As stated in Section 3, ACORN produces tab-delimited ASCII files for a set of ACIS MSIDs. ACORN constantly monitors a given UDP port for spacecraft telemetry. When the telemetry is flowing, i.e. when the OCC has established a communication contact with the CXO, data from these MSIDs are appended to various ASCII files. When the OCC is not in contact with the CXO, these ASCII files are not modified and contain data from the last communication pass.

Three PERL scripts were written that facilitate the monitoring process. One PERL script (acis-read.pl) reads the ACIS ACORN log files and calculates an average and sigma (based on 10 samples of data) for each MSID that is followed. These values and timestamps are then written to another tab-delimited ASCII file. The second PERL script (acis-www.pl) reads these log files and produces an HTML file that displays this information for team members to inspect while at work, home, or on travel (see http://cxc.harvard.edu/acis). The HTML headers instruct the user’s browser to ask for a new version of the page once every 150 seconds. This second PERL script also incorporates the instrument “red” and “yellow” limits discussed in the preceding section. If a given MSID deviates from its nominal value such that it falls within the “red” or “yellow” regime, the value for that MSID is either color-coded “red” or “yellow” on the web page. A third PERL script (limitpager\_v1-4.pl) also reads the ASCII log files produced by acis-read.pl. It compares the average value for each MSID reported against its database of “red” and “yellow” limits. If a MSID breaches its nominal value such that it falls within the “red” or “yellow” value span, a pager alert (for “red limit” violations) or an email alert (for “yellow limit” violations) is issued by the script to key ACIS personnel so that they are immediately notified and so that corrective actions may be taken if required. To reduce the probability of false alerts due to either spurious data or a “noisy” communication contact, the data violation must persist for at least 3 samples before an alert is dispatched.

These three PERL scripts are executed every 5 minutes via a CRON task maintained by a general group UNIX account. This way the thorny issue of permission and read/write priviledges is eliminated. When the OCC is in communication with the CXO, the web page produced by acis-www.pl is updated with fresh values. When the OCC is not in communication with the CXO, acis-www.pl reproduces the last values recorded from the previous communication contact. Since the beginning and end timestamp for each MSID value that went into calculating an average and sigma for that MSID is also presented on the web page, the reader is able to determine whether the displayed value is representative of the current instrument status or whether it is an “old” value from the preceding pass.

This web page, the ACIS Engineering Data Web Page, is a key element in monitoring the health and safety status of the ACIS instrument, and is the primary means by which ACIS anomalies may be identified. Since this web page is world-accessible, it allows key personnel of the CXC to monitor the instrument status whether they are at their office, at home, or even on travel as long as they have internet access. In Section 5.1.1, we provide examples of what was discussed in this section.

#### 5.1.1 Examples

In determining the health and safety limits, it was expected that certain MSIDs would violate their nominal operating range for certain well-known cases. For instance, it is expected that the cold radiators on sides A and B (MSID: 1CRAT and 1CRBT, respectively) would enter its “yellow regime” during perigee transit at certain times of the year as the instrument tends to warm-up during this point in the Chandra orbit. Since the “red” and “yellow” limits are designed to bracket this MSID’s nominal operating temperature range, we expect a violation to occur at perigee transit. Please see Figure 3 for an example of what this alert looks like. Nevertheless, since this is a known condition and since science operation is suspended when the CXO enters the Van Allen radiation belts, the email alerts that would be spawned by limitpager\_v1-4.pl due to “yellow” violations by 1CRAT and 1CBAT are now suppressed when Chandra is entering, at, or exiting from perigee transit.

Secondly, as was mentioned in Section 2.2, the spectral resolution of the front-side CCDs was found to have been significantly reduced early in the mission. It is believed that the primary mechanism for this damage is the result of 100-200 keV protons scattering off Chandra’s mirrors and impacting the CCDs while ACIS was in the focal plane during perigee transit very early in the mission.,, To prevent further damage to the ACIS CCDs, a prevention scheme was implemented by the Science Operations Team of the CXC. One aspect of this scheme has been to limit the orbital proton fluence and flux as measured by the proton monitors on-board the ACE and GOES-8 satellites. One way in which the low energy proton populations can become highly elevated occurs when the Sun unleashes a coronal mass ejection (CME). Once the orbital fluence has reached its threshold value, or if one of the EPHIN channels exceeds its threshold value, Chandra science may be suspended by ground intervention (in the case of the former) or autonomously by the spacecraft (in the case of the latter). When this occurs, the ACIS front-end processors are powered down and the Digital Processing Assembly’s (DPA) input currents decrease such that a “yellow” violation occurs for 1DPICACU and 1DPICBCU. The email alerts generated by this violation serve as a confirmation that Chandra has indeed suspended science operations because of such an event. Figure 4 illustrates what this email alert looks like when 1DPICACU has violated its “yellow” limit. Since the ACIS Engineering Data web page makes extensive use of color and includes various images, an example is not included in this paper (although the URL is presented in Figures 3 and 4).

#### 5.1.2 Peripheral Issues

Lastly, there are some peripheral issues that need to be addressed in order to demonstrate the robustness of the health and safety monitoring. The first issue is one of redundancy. If the machine that hosts the CRON task were to shut down either because of an electrical power failure or due to problems local to that host, the scripts would not run, the web page would not be produced, and the limit-checking would not be performed. To solve this problem, we run these PERL scripts and produce the ACIS Engineering Data web page on two different hosts so that we are not prone to a single-point failure. One host, which produces and performs the “primary” web page and limit-checking, is part of a “mission-critical” suite of SUN work-stations. Should this machine go down for any reason, at any time of the day, the Smithsonian Astrophysical Observatory’s computer system group is tasked with the responsibility of solving whatever problem may afflict the host as soon as possible. The second host, the “back-up”, is run on an ACIS group SUN work-station. Secondly, two additional PERL scripts have been written that deletes old, obsolete ACORN files as well as ensuring that the ACORN process is active on the CPU. If the ACORN process is not found on the CPU, another process is launched. Both of these PERL scripts are also a part of the CRON task that runs on both hosts. Since these have been the primary ways in which the web page may not have been always up-to-date thus far in the mission, we believe we have now developed a scheme of PERL scripts that is robust to a single-point failure and is one that can be relied upon to monitor the health and safety of the ACIS at all times.

### 5.2 Monitoring the ACIS Flight Software Science Telemetry

The second method by which the health and safety status of the ACIS instrument is monitored each and every DSN communication pass is via the ACIS Real-Time Process Monitor (PMON).

The ACIS PMON also receives Chandra telemetry during each real-time contact via a UDP port. A series of shell scripts has been written that decommutates the raw telemetry, extracts ACIS science fields, and produces an HTML file that displays these data. This HTML file presents information that is relevant to the ACIS science run currently in progress. Information such as which CCDs are in use, the number of x-ray “events” found in a given exposure frame, the overclock values for each node amplifier on a given CCD, etc., are displayed. A PMON HTML example is presented in Figure 5.

When PMON receives fresh telemetry, it writes an HTML file every 30 seconds. Like the ACIS Engineering Data web page, the headers in the PMON HTML page instruct the user’s browser to ask for a new version of the page once every 30 seconds. However, when not in real-time contact, or in a non-ACIS telemetry format, some of the displayed values may be very out-of-date. The user, normally ACIS operations personnel, must inspect the relevant time fields to determine whether the data are still useful. As is the case for the scripts used in monitoring the ACIS house-keeping electronics and temperatures, a PERL script has been written that checks to see if the PMON process is active on the CPU. If it is not, another PMON process is launched. This PERL script is run every 5 mins via another CRON task.

Over the course of three years of successful flight operations, there have been three issues that the ACIS Operations team (combining personnel from the CXC as well as the MIT/IPI team) have had to solve with regards to issues of ACIS science operation. One, on a few seemingly random occassions, the ACIS bias maps have been interleaved with ACIS science data. The effect of which is to compromise the science data for that particular CCD and unless corrected, the condition will persist. Since this anomaly was diagnosed, changes to the way in which ACIS is commanded via the weekly load have been made such that we have not seen a repeat of this anomaly, the cause of which is still unknown. The second issue is one where the bias map reports a large number of parity errors. The cause of this anomaly is also not known, however, we have not seen this condition since November of 1999 and it may have been the result of a single-event upset. The last issue is one in which a fatal error is reported by the back-end processor on ACIS. This causes software patches to the ACIS flight software to be lost from memory and requires ground intervention by the ACIS Operations team to resolve. Since all three of these anomalies can be determined via the ACIS PMON, a shell script was written that spawns a pager alert (since immediate attention is required) if one of these anomalies were to occur during a real-time contact . An example of this type of alert is presented in Figure 6 and a comment announcing one of these errors can also be seen on the PMON HTML page (the third line from the top in Figure 5).

## 6 Conclusions

The ACIS instrument on-board the Chandra X-ray Observatory has returned some of the most exquisite, dramatic, and detailed images of the x-ray universe ever seen. The information returned, and the knowledge gained from these ACIS observations, has allowed us to explore another wavelength in rich detail that will undoubtedly propel astronomy forward in this new millenium. However, the continued success of Chandra, and ACIS in particular, is dependent upon diligent operation of the spacecraft as well as the prompt notification, and hopefully resolution, of any anomalies encountered in-flight. In this paper, we have presented two distinct methods by which the telemetry from ACIS during each and every real-time contact is monitored, and in the event of an anomaly, how alerts are quickly dispatched to key ACIS personnel. As new anomalies are discovered in-flight, we will endeavour to find ways to incorporate their monitoring within our current scheme. After three years of highly successful flight operations, we believe we have developed a very robust and reliable suite of PERL and HTML scripts that will help ensure ACIS’ continued success.

## ACKNOWLEDGMENTS

We are grateful to many people, particularly members of the ACIS Operations team, for their support, encouragement, fruitful discussions, and suggestions. We thank all of the engineers, technicians, and scientists who have made the Chandra X-ray Observatory such an incredible success. The authors acknowledge support for this research from NASA contract NAS8-39073.