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Chronic cranial windows have been instrumental in advancing optical studies in vivo, permitting long-term, high-resolution imaging in various brain regions. However, once a window is attached it is difficult to regain access to the brain under the window for cellular manipulations. Here we describe a simple device that combines long term in vivo optical imaging with direct brain access via glass or quartz pipettes and metal, glass, or quartz electrodes for cellular manipulations like dye or drug injections and electrophysiological stimulations or recordings while keeping the craniotomy sterile. Our device comprises a regular cranial window glass coverslip with a drilled access hole later sealed with biocompatible silicone. This chronic cranial window with access port is cheap, easy to manufacture, can be mounted just as the regular chronic cranial window, and is self-sealing after retraction of the pipette or electrode. We demonstrate that multiple injections can be performed through the silicone port by repetitively bolus loading calcium sensitive dye into mouse barrel cortex and recording spontaneous cellular activity over a period of weeks. As an example to the extent of its utility for electrophysiological recording, we describe how simple removal of the silicone seal can permit patch pipette access for whole-cell patch clamp recordings in vivo. During these chronic experiments we do not observe any infections under the window or impairment of animal health.

There is growing interest in understanding calcium dynamics in dendrites, both experimentally and computationally. Many processes influence these dynamics, but in dendrites there is a strong contribution of morphology because the peak calcium levels are strongly determined by the surface to volume ratio of each branch, which is inversely related to branch diameter. In this study we explore the predicted variance of dendritic calcium concentrations due to local changes in dendrite diameter and how this is affected by the modeling approach used. We investigate this in a model of dendritic calcium spiking in different reconstructions of cerebellar Purkinje cells and in morphological analysis of neocortical and hippocampal pyramidal neurons. We report that many published models neglect diameter-dependent effects on calcium concentration and show how to implement this correctly in the NEURON simulator, both for phenomenological pool based models and for implementations using radial 1D diffusion. More detailed modeling requires simulation of 3D diffusion and we demonstrate that this does not dissipate the local concentration variance due to changes of dendritic diameter. In many cases 1D diffusion of models of calcium buffering give a good approximation provided an increased morphological resolution is implemented.

Recording of identified neuronal network activity using genetically encoded calcium indicators (GECIs) requires labeling that is cell type-specific and bright enough for the detection of functional signals. However, specificity and strong expression are often not achievable using the same promoter. Here we present a combinatorial approach for targeted expression and single-cell-level quantification in which a weak promoter is used to drive trans-amplification under a strong general promoter. We demonstrated this approach using recombinant adeno-associated viruses (rAAVs) to deliver the sequence of the GECI D3cpv in the mouse cerebellar cortex. Direct expression under the human synapsin promoter (hSYN) led to high levels of expression (50-100 µM) in five interneuron types of the cerebellar cortex but not in Purkinje cells (PCs) (≤10 μM), yielding sufficient contrast to allow functional signals to be recorded from somata and processes in awake animals using two-photon microscopy. When the hSYN promoter was used to drive expression of the tetracycline transactivator (tTA), a second rAAV containing the bidirectional TET promoter (Ptetbi) could drive strong D3cpv expression in PCs (10-300 µM), enough to allow reliable complex spike detection in the dendritic arbor. An amplified approach should be of use in monitoring neural processing in selected cell types and boosting expression of optogenetic probes. Additionally, we overcome cell toxicity associated with rAAV injection and/or local GECI overexpression by combining the virus injection with systemic pre-injection of hyperosmotic D-mannitol, and by this double the time window for functional imaging.