Whole-cortex in situ sequencing reveals input-dependent area identity.

The cerebral cortex is composed of neuronal types with diverse gene expression that are organized into specialized cortical areas. These areas, each with characteristic cytoarchitecture1,2, connectivity3,4 and neuronal activity5,6, are wired into modular networks3,4,7. However, it remains unclear whether these spatial organizations are reflected in neuronal transcriptomic signatures and how such signatures are established in development. Here we used BARseq, a high-throughput in situ sequencing technique, to interrogate the expression of 104 cell-type marker genes in 10.3 million cells, including 4,194,658 cortical neurons over nine mouse forebrain hemispheres, at cellular resolution. De novo clustering of gene expression in single neurons revealed transcriptomic types consistent with previous single-cell RNA sequencing studies8,9. The composition of transcriptomic types is highly predictive of cortical area identity. Moreover, areas with similar compositions of transcriptomic types, which we defined as cortical modules, overlap with areas that are highly connected, suggesting that the same modular organization is reflected in both transcriptomic signatures and connectivity. To explore how the transcriptomic profiles of cortical neurons depend on development, we assessed cell-type distributions after neonatal binocular enucleation. Notably, binocular enucleation caused the shifting of the cell-type compositional profiles of visual areas towards neighbouring cortical areas within the same module, suggesting that peripheral inputs sharpen the distinct transcriptomic identities of areas within cortical modules. Enabled by the high throughput, low cost and reproducibility of BARseq, our study provides a proof of principle for the use of large-scale in situ sequencing to both reveal brain-wide molecular architecture and understand its development.

Early retinal deprivation crossmodally alters nascent subplate circuits and activity in the auditory cortex during the precritical period.

Sensory perturbation in one modality results in the adaptive reorganization of neural pathways within the spared modalities, a phenomenon known as "crossmodal plasticity," which has been examined during or after the classic "critical period." Because peripheral perturbations can alter the auditory cortex (ACX) activity and functional connectivity of the ACX subplate neurons (SPNs) even before the critical period, called the precritical period, we investigated if retinal deprivation at birth crossmodally alters the ACX activity and SPN circuits during the precritical period. We deprived newborn mice of visual inputs after birth by performing bilateral enucleation. We performed in vivo widefield imaging in the ACX of awake pups during the first two postnatal weeks to investigate cortical activity. We found that enucleation alters spontaneous and sound-evoked activities in the ACX in an age-dependent manner. Next, we performed whole-cell patch clamp recording combined with laser scanning photostimulation in ACX slices to investigate circuit changes in SPNs. We found that enucleation alters the intracortical inhibitory circuits impinging on SPNs, shifting the excitation-inhibition balance toward excitation and this shift persists after ear opening. Together, our results indicate that crossmodal functional changes exist in the developing sensory cortices at early ages before the onset of the classic critical period.

Early retinal deprivation crossmodally alters nascent subplate circuits and activity in the auditory cortex during the precritical period.

Sensory perturbation in one modality results in adaptive reorganization of neural pathways within the spared modalities, a phenomenon known as "crossmodal plasticity", which has been examined during or after the classic 'critical period'. Because peripheral perturbations can alter auditory cortex (ACX) activity and functional connectivity of the ACX subplate neurons (SPNs) even before the classic critical period, called the precritical period, we investigated if retinal deprivation at birth crossmodally alters ACX activity and SPN circuits during the precritical period. We deprived newborn mice of visual inputs after birth by performing bilateral enucleation. We performed in vivo imaging in the ACX of awake pups during the first two postnatal weeks to investigate cortical activity. We found that enucleation alters spontaneous and sound-evoked activity in the ACX in an age-dependent manner. Next, we performed whole-cell patch clamp recording combined with laser scanning photostimulation in ACX slices to investigate circuit changes in SPNs. We found that enucleation alters the intracortical inhibitory circuits impinging on SPNs shifting the excitation-inhibition balance towards excitation and this shift persists after ear opening. Together, our results indicate that crossmodal functional changes exist in the developing sensory cortices at early ages before the onset of the classic critical period.

Cardiac autonomic neuropathy in fibromyalgia: Revisited.

BACKGROUND: Fibromyalgia (FM) is associated with widespread autonomic dysfunction where sympathetic predominance explains associated complaints such as widespread pain, sleep disorders and anxiety. Recent studies indicate a possible neurovascular autonomic interaction in the pathogenesis of FM. OBJECTIVE: Our study paradigm included a modified Ewing's battery of autonomic function tests to find the cardiac autonomic neuropathy (CAN) in FM patients. The battery comprises some tests such as the Valsalva maneuver, which are effort-dependent, so we also aimed to identify a potential simplified test out of the whole battery as an index marker of CAN in FM patients. METHODS: Forty-two female patients with FM were included in this study and were administered sympathetic and parasympathetic reactivity tests to explore the presence of CAN. We compared the results from each sympathetic and parasympathetic reactivity test against CAN. RESULTS: Delta heart rate in the deep breathing test was significantly different in patients with and without CAN. Delta heart rate also exhibited best diagnostic performance (AUC = 0.769, 95% CI: 0.619-0.920, p< 0.001), with 88% sensitivity, 64% specificity, and 89% negative predictive value (NPV). The 30: 15 ratio during the lying to standing test also emerged as a suitable index; however it did not show any difference between CAN and non-CAN patients. CONCLUSION: The delta heart rate has the best diagnostic accuracy, primarily in CAN's exclusion by its very high sensitivity and NPV.

Impaired Hearing and Altered Subplate Circuits During the First and Second Postnatal Weeks of Otoferlin-Deficient Mice.

Sensory deprivation from the periphery impacts cortical development. Otoferlin deficiency leads to impaired cochlear synaptic transmission and is associated with progressive hearing loss in adults. However, it remains elusive how sensory deprivation due to otoferlin deficiency impacts the early development of the auditory cortex (ACX) especially before the onset of low threshold hearing. To test that, we performed in vivo imaging of the ACX in awake mice lacking otoferlin (Otof-/-) during the first and second postnatal weeks and found that spontaneous and sound-driven cortical activity were progressively impaired. We then characterized the effects on developing auditory cortical circuits by performing in vitro recordings from subplate neurons (SPN), the first primary targets of thalamocortical inputs. We found that in Otof-/- pups, SPNs received exuberant connections from excitatory and inhibitory neurons. Moreover, as a population, SPNs showed higher similarity with respect to their circuit topology in the absence of otoferlin. Together, our results show that otoferlin deficiency results in impaired hearing and has a powerful influence on cortical connections and spontaneous activity in early development even before complete deafness. Therefore, peripheral activity has the potential to sculpt cortical structures from the earliest ages, even before hearing impairment is diagnosed.

Changing subplate circuits: Early activity dependent circuit plasticity.

Early neural activity in the developing sensory system comprises spontaneous bursts of patterned activity, which is fundamental for sculpting and refinement of immature cortical connections. The crude early connections that are initially refined by spontaneous activity, are further elaborated by sensory-driven activity from the periphery such that orderly and mature connections are established for the proper functioning of the cortices. Subplate neurons (SPNs) are one of the first-born mature neurons that are transiently present during early development, the period of heightened activity-dependent plasticity. SPNs are well integrated within the developing sensory cortices. Their structural and functional properties such as relative mature intrinsic membrane properties, heightened connectivity via chemical and electrical synapses, robust activation by neuromodulatory inputs-place them in an ideal position to serve as crucial elements in monitoring and regulating spontaneous endogenous network activity. Moreover, SPNs are the earliest substrates to receive early sensory-driven activity from the periphery and are involved in its modulation, amplification, and transmission before the maturation of the direct adult-like thalamocortical connectivity. Consequently, SPNs are vulnerable to sensory manipulations in the periphery. A broad range of early sensory deprivations alters SPN circuit organization and functions that might be associated with long term neurodevelopmental and psychiatric disorders. Here we provide a comprehensive overview of SPN function in activity-dependent development during early life and integrate recent findings on the impact of early sensory deprivation on SPNs that could eventually lead to neurodevelopmental disorders.

Early peripheral activity alters nascent subplate circuits in the auditory cortex.

Cortical function can be shaped by sensory experience during a critical period. The onset of the critical period is thought to coincide with the onset of thalamocortical transmission to the thalamo-recipient layer 4 (L4). In early development, subplate neurons (SPNs), and not L4 neurons, are the first targets of thalamic afferents. SPNs are transiently involved in early development and are largely eliminated during development. Activation of L4 by thalamic afferents coincides with the opening of ear canal (~P11 in mice) and precedes the later critical period. Here, we show in mice that abolishing peripheral function or presenting sound stimuli even before P11 leads to bidirectionally altered functional connectivity of SPNs in auditory cortex. Thus, early sensory experience can sculpt subplate circuits before thalamocortical circuits to L4 are mature. Our results show that peripheral activity shapes cortical circuits in a sequential manner and from earlier ages than has been appreciated.

Corollary discharge in precerebellar nuclei of sleeping infant rats.

In week-old rats, somatosensory input arises predominantly from external stimuli or from sensory feedback (reafference) associated with myoclonic twitches during active sleep. A previous study suggested that the brainstem motor structures that produce twitches also send motor copies (or corollary discharge, CD) to the cerebellum. We tested this possibility by recording from two precerebellar nuclei-the inferior olive (IO) and lateral reticular nucleus (LRN). In most IO and LRN neurons, twitch-related activity peaked sharply around twitch onset, consistent with CD. Next, we identified twitch-production areas in the midbrain that project independently to the IO and LRN. Finally, we blocked calcium-activated slow potassium (SK) channels in the IO to explain how broadly tuned brainstem motor signals can be transformed into precise CD signals. We conclude that the precerebellar nuclei convey a diversity of sleep-related neural activity to the developing cerebellum to enable processing of convergent input from CD and reafferent signals.

Wakefulness suppresses retinal wave-related neural activity in visual cortex.

In the developing visual system before eye opening, spontaneous retinal waves trigger bursts of neural activity in downstream structures, including visual cortex. At the same ages when retinal waves provide the predominant input to the visual system, sleep is the predominant behavioral state. However, the interactions between behavioral state and retinal wave-driven activity have never been explicitly examined. Here we characterized unit activity in visual cortex during spontaneous sleep-wake cycles in 9- and 12-day-old rats. At both ages, cortical activity occurred in discrete rhythmic bursts, ~30-60 s apart, mirroring the timing of retinal waves. Interestingly, when pups spontaneously woke up and moved their limbs in the midst of a cortical burst, the activity was suppressed. Finally, experimentally evoked arousals also suppressed intraburst cortical activity. All together, these results indicate that active wake interferes with the activation of the developing visual cortex by retinal waves. They also suggest that sleep-wake processes can modulate visual cortical plasticity at earlier ages than has been previously considered.NEW & NOTEWORTHY By recording in visual cortex in unanesthetized infant rats, we show that neural activity attributable to retinal waves is specifically suppressed when pups spontaneously awaken or are experimentally aroused. These findings suggest that the relatively abundant sleep of early development plays a permissive functional role for the visual system. It follows, then, that biological or environmental factors that disrupt sleep may interfere with the development of these neural networks.

Twitch-related and rhythmic activation of the developing cerebellar cortex.

The cerebellum is a critical sensorimotor structure that exhibits protracted postnatal development in mammals. Many aspects of cerebellar circuit development are activity dependent, but little is known about the nature and sources of the activity. Based on previous findings in 6-day-old rats, we proposed that myoclonic twitches, the spontaneous movements that occur exclusively during active sleep (AS), provide generalized as well as topographically precise activity to the developing cerebellum. Taking advantage of known stages of cerebellar cortical development, we examined the relationship between Purkinje cell activity (including complex and simple spikes), nuchal and hindlimb EMG activity, and behavioral state in unanesthetized 4-, 8-, and 12-day-old rats. AS-dependent increases in complex and simple spike activity peaked at 8 days of age, with 60% of units exhibiting significantly more activity during AS than wakefulness. Also, at all three ages, approximately one-third of complex and simple spikes significantly increased their activity within 100 ms of twitches in one of the two muscles from which we recorded. Finally, we observed rhythmicity of complex and simple spikes that was especially prominent at 8 days of age and was greatly diminished by 12 days of age, likely due to developmental changes in climbing fiber and mossy fiber innervation patterns. All together, these results indicate that the neurophysiological activity of the developing cerebellum can be used to make inferences about changes in its microcircuitry. They also support the hypothesis that sleep-related twitches are a prominent source of discrete climbing and mossy fiber activity that could contribute to the activity-dependent development of this critical sensorimotor structure.

Glutamate microinjection in the medial septum of rats decreases paradoxical sleep and increases slow wave sleep.

The role of the medial septum in suppressing paradoxical sleep and promoting slow wave sleep was suggested on the basis of neurotoxic lesion studies. However, these conclusions need to be substantiated with further experiments, including chemical stimulation studies. In this report, the medial septum was stimulated in adult male rats by microinjection of L-glutamate. Sleep-wakefulness was electrophysiologically recorded, through chronically implanted electrodes, for 2 h before the injection and 4 h after the injection. There was a decrease in paradoxical sleep during the first hour and an increase in slow wave sleep during the second hour after the injection. The present findings not only supported the lesion studies but also showed that the major role of the medial septum is to suppress paradoxical sleep.