Diversity in the structure of action potential‐mediated neural connectivity within rat supra chiasmatic nucleus

Abstract

Action potential (AP)‐mediated cell‐to‐cell communication is essential for the frequency‐locking and phase‐synchronization of the clock cells within the biological master clock, suprachiasmatic nucleus (SCN). Nevertheless, the morphology of its network connectivity is largely unexplored. Here, with an optimized optogenetic light‐stimulation and scanning protocol, we report some key characteristics of the inhibitory receptive field (IRF), the area which brings inhibitory synaptic currents to a given target cell, and basic statistics of the inhibitory network connections of rat SCN clock cells. ChR2 transfected, slice cultures of rat SCN were stimulated by a blue power LED light in a repetitive box‐scanning modes, while a target cell was whole‐cell patched. The registered inhibitory postsynaptic currents, which were brought by light‐induced APs of presynaptic neurons, were mostly GABAergic. The sizes and shapes of IRFs of SCN cells were very diverse, and the number of presynaptic cells making up the IRF of a given target cell followed an exponential distribution with an average value of 8.9 approximately, according to our clustering analysis which is based on a hybrid measure D, combining the physical distance r and the difference in the current amplitudes of two different sites. Although this estimate inevitably depends on the construct of the measure D, it is found not so sensitive on the parameter w, which weighs the relative significance of the current amplitude different with respect to the physical distance r: The average number of presynaptic neurons varies < 26% over a significant range of 0 < w < 30. On average, the presynaptic connection number density around a target cell falls off as an exponentially decreasing function of r. But, its space constant (~210.7 μm) is quite large that long‐range (>210.7 μm) neural connections are abundant (>66.9%) within the SCN.

Deciphering clock cell network morphology within the biological master clock, suprachiasmatic nucleus: From the perspective of circadian wave dynamics

“…In addition to the sparseness, Fan et al .’s analysis also suggested that afferent VIP+ connections are biased toward a subset of neurons. About the same time we also carried out an optogenetic connectome study of our own using a different experimental platform and protocol on slice cultures of neonatal rat SCNs to obtain two important statistics concerning the SCN network morphology [ 13 ]: the number of afferent connections and the distances to presynaptic clock cells projecting to a postsynaptic clock cell. With the statistics, we estimated the average indegree k in per each clock cell to be 8.9, a number considerably larger than the estimated value of 0.66 based on the work of Fan et al .…”

“…As far as we know, these two reports are the only cases directly looking at the clock cell network connectivity within the SCN based on an optogenetic mapping technique. In fact, the optogenetic connectome study probing the SCN network connectivity is technically very challenging and extremely time demanding [ 13 ]. In addition, the mapping technique inherently cannot give any information about efferent connections (eg, outdegree k out ).…”

“…Earlier, we (HK, CM, and KJL) carried out optogenetic connectome mapping analysis on slice cultures of rat SCN to characterize clock-cell network connections [ 13 ]. Briefly, organotypic SCN slice cultures were prepared using postnatal 3–5 days old Sprague-Dawley rats following a standard procedure as described in our previous paper [ 13 ]. After brain slices were cultured for about a week, ChR2 AVV virus (rAAV2/hsyn-hsyn-hchR2 (H134R)—EYFP-WPRE-PA) was injected for ChR2 transfection.…”

“…So, for example, when the degree correlation is positive (negative), the larger (smaller) k in a node has, the larger (smaller) k out it will have. Since the indegrees of the SCN clock cells in the earlier experiment were found to follow an exponential distribution with an average value of 8.9 [ 13 ], we randomly assign an indegree for each of all 5,000 model cells to follow the same exponential distribution. For generating our archive of model SCN networks, we have considered 5 (3) different positive (negative) k in − k out correlation levels and 1 randomly correlated case, which are labeled by pos(10), pos(20), pos(30), pos(40), pos(50), neg(10), neg(20, neg(30), and none(0), respectively (Sec.…”

“…As far as we know, however, there is no direct experimental confirmation about it, and here we have explored the following two different possibilities: 1) The SCN clock cells in the core region have a higher degree than that of the shell region or 2) the opposite case. Again, our model SCN is a 2D square-grid mesh shaped in the actual coronal section of a rat SCN slice culture [ 13 ], as illustrated in Fig 4A . We can systematically arrange the distance of a node from the center (star marks in Fig 4A ) according to its value of k total , k in or k out (see S1 Text ).…”