Neonatal Hypoxia-Ischemia Causes Persistent Intracortical Circuit Changes in Layer 4 of Rat Auditory Cortex.

The connection between early brain injury and subsequent development of disorders is unknown. Neonatal hypoxia-ischemia (HI) alters circuits associated with subplate neurons (SPNs). SPNs are among the first maturing cortical neurons, project to thalamorecipient layer 4 (L4), and are required for the development of thalamocortical connections. Thus, early HI might influence L4 and such influence might persist. We investigated functional circuits to L4 neurons in neonatal rat HI models of different severities (mild and moderate) shortly after injury and at adolescence. We used laser-scanning photostimulation in slices of auditory cortex during P5-10 and P18-23. Mild injuries did not initially (P6/P7) alter the convergence of excitatory inputs from L2/3, but hyperconnectivity emerged by P8-10. Inputs from L4 showed initial hypoconnectivity which resolved by P8-10. Moderate injuries resulted in initial hypoconnectivity from both layers which resolved by P8-10 and led to persistent strengthening of connections. Inhibitory inputs to L4 cells showed similar changes. Functional changes were mirrored by reduced dendritic complexity. We also observed a persistent increase in similarity of L4 circuits, suggesting that HI interferes with developmental circuit refinement and diversification. Altogether, our results show that neonatal HI injuries lead to persistent changes in intracortical connections.

Transient Subgranular Hyperconnectivity to L2/3 and Enhanced Pairwise Correlations During the Critical Period in the Mouse Auditory Cortex.

During the critical period, neuronal connections are shaped by sensory experience. While the basis for this temporarily heightened plasticity remains unclear, shared connections introducing activity correlations likely play a key role. Thus, we investigated the changing intracortical connectivity in primary auditory cortex (A1) over development. In adult, layer 2/3 (L2/3) neurons receive ascending inputs from layer 4 (L4) and also receive few inputs from subgranular layer 5/6 (L5/6). We measured the spatial pattern of intracortical excitatory and inhibitory connections to L2/3 neurons in slices of mouse A1 across development using laser-scanning photostimulation. Before P11, L2/3 cells receive most excitatory input from within L2/3. Excitatory inputs from L2/3 and L4 increase after P5 and peak during P9-16. L5/6 inputs increase after P5 and provide most input during P12-16, the peak of the critical period. Inhibitory inputs followed a similar pattern. Functional circuit diversity in L2/3 emerges after P16. In vivo two-photon imaging shows low pairwise signal correlations in neighboring neurons before P11, which peak at P15-16 and decline after. Our results suggest that the critical period is characterized by high pairwise activity correlations and that transient hyperconnectivity of specific circuits, in particular those originating in L5/6, might play a key role.

Neonatal Hypoxia-Ischemia Causes Functional Circuit Changes in Subplate Neurons.

Neonatal hypoxia-ischemia (HI) in the preterm human results in damage to subcortical developing white matter and cognitive impairments. Subplate neurons (SPNs) are among the first-born cortical neurons and are necessary for normal cerebral development. While moderate or severe HI at P1 in rats leads to SPN loss, it is unclear if HI, esp. forms not associated with overt cell loss lead to altered SPN circuits. Thus, we used two HI models with different severities in P1 rats. Cauterization of the common carotid artery (CCA) causes a largely transient and thus milder ischemia (HI-Caut) while CCA ligation causes more severe ischemia (HI-Lig). While HI-Lig caused subplate damage, HI-Caut did not cause overt histological damage on the light microscopic level. We used laser-scanning photostimulation (LSPS) in acute thalamocortical slices of auditory cortex during P5-10 to study the functional connectivity of SPNs. Both HI categories resulted in hyperconnectivity of excitatory and inhibitory circuits to SPNs. Thus, alterations on the circuit level are present in the absence of cell loss. Our results show that SPN circuits are uniquely susceptible to HI. Given the key developmental role of SPNs, our results suggest that altered SPN circuits might underlie the abnormal development of cortical function after HI.

Molecularly Defined Subplate Neurons Project Both to Thalamocortical Recipient Layers and Thalamus.

In mammals, subplate neurons (SPNs) are among the first generated cortical neurons. While most SPNs exist only transiently during development, a number of SPNs persist among adult Layer 6b (L6b). During development, SPNs receive thalamic and intra-cortical input, and primarily project to Layer 4 (L4). SPNs are critical for the anatomical and functional development of thalamocortical connections and also pioneer corticothalamic projections. Since SPNs are heterogeneous, SPN subpopulations might serve different roles. Here, we investigate the connectivity of one subpopulation, complexin-3 (Cplx3)-positive SPNs (Cplx3-SPNs), in mouse whisker somatosensory (barrel) cortex (S1). We find that many Cplx3-SPNs survive into adulthood and become a subpopulation of L6b. Cplx3-SPNs axons project to thalamorecipient layers, that is, L4, 5a, and 1. The L4 projections are biased towards the septal regions between barrels in the second postnatal week. Thus, S1 Cplx3-SPN targets co-localize with the eventual projections of the medial posterior thalamic nucleus (POm). In addition to their cortical targets, Cplx3-SPNs also extend long-range axons to several thalamic nuclei, including POm. Thus, Cplx3-SPN/L6b neurons are associated with paralemniscal pathways and can potentially directly link thalamocortical and corticothalamic circuits. This suggests an additional key role for SPNs in the establishment and maintenance of thalamocortical processing.

Mild systemic inflammation and moderate hypoxia transiently alter neuronal excitability in mouse somatosensory cortex.

During the perinatal period, the brain is highly vulnerable to hypoxia and inflammation, which often cause white matter injury and long-term neuronal dysfunction such as motor and cognitive deficits or epileptic seizures. We studied the effects of moderate hypoxia (HYPO), mild systemic inflammation (INFL), or the combination of both (HYPO+INFL) in mouse somatosensory cortex induced during the first postnatal week on network activity and compared it to activity in SHAM control animals. By performing in vitro electrophysiological recordings with multi-electrode arrays from slices prepared directly after injury (P8-10), one week after injury (P13-16), or in young adults (P28-30), we investigated how the neocortical network developed following these insults. No significant difference was observed between the four groups in an extracellular solution close to physiological conditions. In extracellular 8mM potassium solution, slices from the HYPO, INFL, and HYPO+INFL group were more excitable than SHAM at P8-10 and P13-16. In these two age groups, the number and frequency of spontaneous epileptiform events were significantly increased compared to SHAM. The frequency of epileptiform events was significantly reduced by the NMDA antagonist D-APV in HYPO, INFL, and HYPO+INFL, but not in SHAM, indicating a contribution of NMDA receptors to this pathophysiological activity. In addition, the AMPA/kainate receptor antagonist CNQX suppressed the remaining epileptiform activity. Electrical stimulation evoked prominent epileptiform activity in slices from HYPO, INFL and HYPO+INFL animals. Stimulation threshold to elicit epileptiform events was lower in these groups than in SHAM. Evoked events spread over larger areas and lasted longer in treated animals than in SHAM. In addition, the evoked epileptiform activity was reduced in the older (P28-30) group indicating that cortical dysfunction induced by hypoxia and inflammation was transient and compensated during early development.

Subplate neurons promote spindle bursts and thalamocortical patterning in the neonatal rat somatosensory cortex.

Patterned neuronal activity such as spindle bursts in the neonatal cortex is likely to promote the maturation of cortical synapses and neuronal circuits. Previous work on cats has shown that removal of subplate neurons, a transient neuronal population in the immature cortex, prevents the functional maturation of thalamocortical and intracortical connectivity. Here we studied the effect of subplate removal in the neonatal rat primary somatosensory cortex (S1). Using intracortical EEG we show that after selective removal of subplate neurons in the limb region of S1, endogenous and sensory evoked spindle burst activity is largely abolished. Consistent with the reduced in vivo activity in the S1 limb region, we find by in vitro recordings that thalamocortical inputs to layer 4 neurons are weak. In addition, we find that removal of subplate neurons in the S1 barrel region prevents the development of the characteristic histological barrel-like appearance. Thus, subplate neurons are crucially involved in the generation of particular types of early network activity in the neonatal cortex, which are an important feature of cortical development. The altered EEG pattern following subplate damage could be applicable in the neurological assessment of human neonates.