Calibration of insulin pumps based on discrete doses at given cycle times.

One application in the medical treatment at very small flow rates is the usage of an Insulin pump that delivers doses of insulin at constant cycle times for a specific basal rate as quasi-continuous insulin delivery, which is an important cornerstone in diabetes management. The calibration of these basal rates are performed by either gravimetric or optical methods, which have been developed within the European Metrology Program for Innovation and Research (EMPIR) Joint Research Project (JRP) 18HLT08 Metrology for drug delivery II (MeDDII). These measurement techniques are described in this paper, and an improved approach of the analytical procedure given in the standard IEC 60601-2-24:2012 for determining the discrete doses and the corresponding basal rates is discussed in detail. These improvements allow detailed follow up of dose cycle time and delivered doses as a function of time to identify some artefacts of the measurement method or malfunctioning of the insulin pump. Moreover, the calibration results of different basal rates and bolus deliveries for the gravimetric and the optical methods are also presented. Some analysis issues that should be addressed to prevent misinterpreting of the calibration results are discussed. One of the main issues is the average over a period of time which is an integer multiple of the cycle time to determine the basal rate with the analytical methods described in this paper.

Probing subicular inputs to the medial prefrontal cortex.

The hippocampal formation is anatomically and functionally divided into a dorsal and a ventral part, being involved in processing cognitive tasks and emotional stimuli, respectively. The ventral subiculum as part of the hippocampal formation projects to the medial prefrontal cortex (mPFC), but only very little is known about connections arising from the dorsal SUB (dSUB). Here, we investigate the dSUB to mPFC connectivity in acute brain slices using electrophysiology and optogenetics. We show that the anterior cingulate cortex (ACC) is the main target of dorsal subicular projections to the mPFC, with no preference between excitatory or inhibitory neurons. In addition to superficial neurons in the ACC, the prelimbic and infralimbic PFC are also targeted by subicular fibers. Thus, these novel region- and layer-specific connections between the dSUB and the prefrontal cortices challenge existing anatomical data and refine the hippocampocortical wiring diagram.

NDNF is selectively expressed by neocortical, but not habenular neurogliaform cells.

The lateral habenula (LHb) is a brain structure which is known to be pathologically hyperactive in depression, whereby it shuts down the brains' reward systems. Interestingly, inhibition of the LHb has been shown to have an antidepressant effect, hence making the LHb a fascinating subject of study for developing novel antidepressant therapies. Despite this however, the exact mechanisms by which inhibitory signalling is processed within the LHb remain incompletely understood. Some studies have proposed the existence of locally targeting inhibitory interneuron populations within the LHb. One such population is believed to be akin to neocortical neurogliaform cells, yet specific molecular markers for studying these neurons are sparse and hence their function remains elusive. Recently, neuron-derived neurotrophic factor (NDNF) has been proposed as one such marker for neocortical neurogliaform cells. Using a combination of histological, physiological and optogenetic tools, we hence sought to first validate if NDNF was selectively expressed by such inhibitory neurons within the neocortex, and then if it was confined to a similar population within the LHb. While we report this to be true for the neocortex, we find no such evidence within the LHb; rather that NDNF is expressed without restriction to a particular neuronal subpopulation. These results hence indicate that molecular markers can represent broadly diverse populations of neurons on a region-to-region basis and that therefore each population as defined by molecular marker expression should be validated in each brain structure.

Imaging Pericytes and the Regulation of Cerebral Blood Flow.

The brain's high energy requirements drive the need for close coupling of local neuronal activity to blood supply. Capillaries have been shown to dilate before arterioles in response to sensory stimulation, pointing to a key role for microvascular pericytes in mediating cerebrovascular dynamics. However, many aspects of these cells' function remain unknown and even controversial, from their identification, to the mechanism and regulation of their contractility in physiology and disease. Investigating how pericytes regulate vascular diameter is therefore likely to be the subject of many future experiments. Here we provide protocols for three different techniques (ex vivo slice imaging, in vivo imaging, and immunohistochemistry) that are highly valuable for performing such experiments.

Disentangling neuronal inhibition and inhibitory pathways in the lateral habenula.

The lateral habenula (LHb) is hyperactive in depression, and thus potentiating inhibition of this structure makes an interesting target for future antidepressant therapies. However, the circuit mechanisms mediating inhibitory signalling within the LHb are not well-known. We addressed this issue by studying LHb neurons expressing either parvalbumin (PV) or somatostatin (SOM), two markers of particular sub-classes of neocortical inhibitory neurons. Here, we find that both PV and SOM are expressed by physiologically distinct sub-classes. Furthermore, we describe multiple sources of inhibitory input to the LHb arising from both local PV-positive neurons, from PV-positive neurons in the medial dorsal thalamic nucleus, and from SOM-positive neurons in the ventral pallidum. These findings hence provide new insight into inhibitory control within the LHb, and highlight that this structure is more neuronally diverse than previously thought.

Correction of z-motion artefacts to allow population imaging of synaptic activity in behaving mice.

KEY POINTS: Motion artefacts associated with motor behaviour are an inevitable problem of multiphoton imaging in awake behaving animals, particularly when imaging synapses. Correction of axial motion artefacts usually requires volumetric imaging resulting in slower rates of acquisition. We describe a method to correct z-motion artefacts that is easy to implement and allows population imaging of synaptic activity while scanning a single plane in a standard multiphoton microscope. The method uses a reference volume acquired in two colour channels - an activity reporter and an anatomical marker of blood vessels. The procedure estimates the z-displacement in every frame and applies an intensity correction in which the z intensity profile for each synapse is modelled as a Moffat function. We demonstrate that the method allows synaptic calcium signals to be collected from populations of synaptic boutons in mouse primary visual cortex during locomotion. ABSTRACT: Functional imaging of head-fixed, behaving mice using two-photon imaging of fluorescent activity reporters has become a powerful tool for studying the function of the brain. Motion artefacts are an inevitable problem during such experiments and are routinely corrected for in x and y dimensions. However, axial (z) shifts of several microns can also occur, leading to intensity fluctuations in structures such as synapses that are small compared to the axial point-spread function of the microscope. Here we present a simple strategy to correct z-motion artefacts arising over the course of a time-series experiment in a single optical plane. Displacement in z was calculated using dye-filled blood vessels as an anatomical marker, providing high contrast images and accuracy to within ∼0.1 µm. The axial profiles of ROIs corresponding to synapses were described using a Moffat function and this 'ROI-spread function' used to correct activity traces on an ROI-by-ROI basis. We demonstrate the accuracy and utility of the procedures in simulation experiments using fluorescent beads and then apply them to correcting measurements of synaptic activity in populations of vasoactive-intestinal peptide (VIP) interneurons expressing the synaptic reporter SyGCaMP6f. Correction of z-motion artefacts had a substantial impact on the apparent correlation between synaptic activity and running speed, demonstrating the importance of correcting these when performing imaging experiments in awake mice.

Feedback-induced glutamate spillover enhances negative feedback from horizontal cells to cones.

KEY POINTS: In the retina, horizontal cells feed back negatively to cone photoreceptors. Glutamate released from cones can spill over to neighbouring cones. Here we show that cone glutamate release induced by negative feedback can also spill over to neighbouring cones. This glutamate activates the glutamate transporter-associated chloride current in these neighbouring cones, which leads to a change in their membrane potential and thus modulates their output. In this way, feedback-induced glutamate spillover enhances negative feedback from horizontal cells to cones, thus forming an additional feedback pathway. This effect will be particularly prominent in cones that are strongly hyperpolarized by light. ABSTRACT: Inhibition in the outer retina functions via an unusual mechanism. When horizontal cells hyperpolarize the activation potential of the Ca(2+) current of cones shifts to more negative potentials. The underlying mechanism consists of an ephaptic component and a Panx1/ATP-mediated component. Here we identified a third feedback component, which remains active outside the operating range of the Ca(2+) current. We show that the glutamate transporters of cones can be activated by glutamate released from their neighbours. This pathway can be triggered by negative feedback from horizontal cells to cones, thus providing an additional feedback pathway. This additional pathway is mediated by a Cl(-) current, can be blocked by either removing the gradient of K(+) or by adding the glutamate transporter blocker TBOA, or low concentrations of Zn(2+) . These features point to a glutamate transporter-associated Cl(-) current. The pathway has a delay of 4.7 ± 1.7 ms. The effectiveness of this pathway in modulating the cone output depends on the equilibrium potential of Cl(-) (ECl ) and the membrane potential of the cone. Because estimates of ECl show that it is around the dark resting membrane potential of cones, the activation of the glutamate transporter-associated Cl(-) current will be most effective in changing the membrane potential during strong hyperpolarization of cones. This means that negative feedback would particularly be enhanced by this pathway when cones are hyperpolarized. Spatially, this pathway does not reach further than the direct neighbouring cones. The consequence is that this feedback pathway transmits information between cones of different spectral type.

Extracellular ATP hydrolysis inhibits synaptic transmission by increasing ph buffering in the synaptic cleft.

Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms), highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca²âº channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form of synaptic modulation may be a widespread phenomenon.

Ephaptic communication in the vertebrate retina.

In the vertebrate retina, cones project to the horizontal cells (HCs) and bipolar cells (BCs). The communication between cones and HCs uses both chemical and ephaptic mechanisms. Cones release glutamate in a Ca(2+)-dependent manner, while HCs feed back to cones via an ephaptic mechanism. Hyperpolarization of HCs leads to an increased current through connexin hemichannels located on the tips of HC dendrites invaginating the cone synaptic terminals. Due to the high resistance of the extracellular synaptic space, this current makes the synaptic cleft slightly negative. The result is that the Ca(2+)-channels in the cone presynaptic membrane experience a slightly depolarized membrane potential and therefore more glutamate is released. This ephaptic mechanism forms a very fast and noise free negative feedback pathway. These characteristics are crucial, since the retina has to perform well in demanding conditions such as low light levels. In this mini-review we will discuss the critical components of such an ephaptic mechanism. Furthermore, we will address the question whether such communication appears in other systems as well and indicate some fundamental features to look for when attempting to identify an ephaptic mechanism.