Epileptic baboons have lower numbers of neurons in specific areas of cortex

Epilepsy is characterized by recurrent seizure activity that can induce pathological reorganization and alter normal function in neocortical networks. In the present study, we determined the numbers of cells and neurons across the complete extent of the cortex for two epileptic baboons with naturally occurring seizures and two baboons without epilepsy. Overall, the two epileptic baboons had a 37% average reduction in the number of cortical neurons compared with the two nonepileptic baboons. The loss of neurons was variable across cortical areas, with the most pronounced loss in the primary motor cortex, especially in lateral primary motor cortex, representing the hand and face. Less-pronounced reductions of neurons were found in other parts of the frontal cortex and in somatosensory cortex, but no reduction was apparent in the primary visual cortex and little in other visual areas. The results provide clear evidence that epilepsy in the baboon is associated with considerable reduction in the numbers of cortical neurons, especially in frontal areas of the cortex related to motor functions. Whether or not the reduction of neurons is a cause or an effect of seizures needs further investigation.Epilepsy is associated with structural changes in the cerebral cortex (e.g., refs. 1–6), and partial epilepsies (i.e., seizures originating from a brain region) may lead to loss of neurons (7) and altered connectivity (8). The cerebral cortex is a heterogeneous structure comprised of multiple sensory and motor information-processing systems (e.g., refs. 9 and 10) that vary according to their processing demands, connectivity (e.g., refs. 11 and 12), and intrinsic numbers of cells and neurons (13–16). Chronic seizures have been associated with progressive changes in the region of the epileptic focus and in remote but functionally connected cortical or subcortical structures (3, 17). Because areas of the cortex are functionally and structurally different, they may also differ in susceptibility to pathological changes resulting from epilepsy.The relationship between seizure activity and neuron damage can be difficult to study in humans. Seizure-induced neuronal damage can be convincingly demonstrated in animals using electrically or chemically induced status epilepticus (one continuous seizure episode longer than 5 min) to reveal morphometric (e.g., refs. 18 and 19) or histological changes (e.g., refs. 20 and 21). Subcortical brain regions are often studied for vulnerability to seizure-induced injury (21–27); however, a recent study by Karbowski et al. (28) observed reduction of neurons in cortical layers 5 and 6 in the frontal lobes of rats with seizures. Seizure-induced neuronal damage in the cortex has also been previously demonstrated in baboons with convulsive status epilepticus (29).The goal of the present study was to determine if there is a specific pattern of cell or neuron reduction across the functionally divided areas of the neocortex in baboons with epilepsy. Selected strains of baboons have been studied as a natural primate model of generalized epilepsy (30–36) that is analogous to juvenile myoclonic epilepsy in humans. The baboons demonstrate generalized myoclonic and tonic-clonic seizures, and they have generalized interictal and ictal epileptic discharges on scalp EEG. Because of their phylogenetic proximity to humans, baboons and other Old World monkeys share many cortical areas and other features of cortical organization with humans (e.g., refs. 9 and 10). Cortical cell and neuron numbers were determined using the flow fractionator method (37, 38) in epileptic baboon tissue obtained from the Texas Biomedical Research Institute, where a number of individuals develop generalized epilepsy within a pedigreed baboon colony (31–36). Our results reveal a regionally specific neuron reduction in the cortex of baboons with naturally occurring, generalized seizures.     

From the Cover: PNAS Plus: Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro

Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro.Induced pluripotent stem cell (iPSC) technology is currently being used to model human diseases in vitro and may contribute to the discovery and validation of new pharmacological treatments (1–3). In particular, neuroscientists have seized the opportunity to culture neurons from patients with neurological and psychiatric disorders and have demonstrated that phenotypes associated with particular disorders can be recapitulated in the dish (4–7). However, the basic culture conditions for growing neurons in vitro have not been updated to reflect fundamental principles of brain physiology. Currently, most human neuronal cultures are grown in media based on DMEM/F12 (4, 5, 7–24), Neurobasal (25–30), or a mixture of DMEM and Neurobasal (DN) (31–34). To promote neuronal differentiation and survival, a variety of supplements, such as serum, growth factors, hormones, proteins, and antioxidants, are typically added to these basal media. Although these media were designed and optimized to promote neuronal survival in vitro, they were not tested for their ability to support fundamental neuronal functions. Using several electrophysiological techniques such as patch clamping, calcium imaging, and multielectrode arrays, we found that widely used tissue culture media (e.g., DMEM basal, Neurobasal, serum) actually impaired neurophysiological functions.We identified several neuroactive components in these media that acutely interfered with neuronal function. To solve these issues, we designed a chemically defined basal medium: BrainPhys basal. We used human neurons in vitro to demonstrate in a series of experiments that this new medium, combined with the appropriate supplements, better supports important neuronal functions while sustaining cell survival in vitro. Notably, BrainPhys-based medium better mimics the environment present in healthy living brains, unlike previous media based on DMEM, Neurobasal or serum. Although BrainPhys basal was specifically designed for the culturing of mature human neurons, our studies also showed that BrainPhys provided a functional environment for ex vivo brain slices and for culturing rodent primary neurons.     

Local generation of multineuronal spike sequences in the hippocampal CA1 region

Sequential activity of multineuronal spiking can be observed during theta and high-frequency ripple oscillations in the hippocampal CA1 region and is linked to experience, but the mechanisms underlying such sequences are unknown. We compared multineuronal spiking during theta oscillations, spontaneous ripples, and focal optically induced high-frequency oscillations (“synthetic” ripples) in freely moving mice. Firing rates and rate modulations of individual neurons, and multineuronal sequences of pyramidal cell and interneuron spiking, were correlated during theta oscillations, spontaneous ripples, and synthetic ripples. Interneuron spiking was crucial for sequence consistency. These results suggest that participation of single neurons and their sequential order in population events are not strictly determined by extrinsic inputs but also influenced by local-circuit properties, including synapses between local neurons and single-neuron biophysics.A hypothesized hallmark of cognition is self-organized sequential activation of neuronal assemblies (1). Self-organized neuronal sequences have been observed in several cortical structures (2–5) and neuronal models (6–7). In the hippocampus, sequential activity of place cells (8) may be induced by external landmarks perceived by the animal during spatial navigation (9) and conveyed to CA1 by the upstream CA3 region or layer 3 of the entorhinal cortex (10). Internally generated sequences have been also described in CA1 during theta oscillations in memory tasks (4, 11), raising the possibility that a given neuronal substrate is responsible for generating sequences at multiple time scales. The extensive recurrent excitatory collateral system of the CA3 region has been postulated to be critical in this process (4, 7, 12, 13).The sequential activity of place cells is “replayed” during sharp waves (SPW) in a temporally compressed form compared with rate modulation of place cells (14–20) and may arise from the CA3 recurrent excitatory networks during immobility and slow wave sleep. The SPW-related convergent depolarization of CA1 neurons gives rise to a local, fast oscillatory event in the CA1 region (“ripple,” 140–180 Hz; refs. 8 and 21). Selective elimination of ripples during or after learning impairs memory performance (22–24), suggesting that SPW ripple-related replay assists memory consolidation (12, 13). Although the local origin of the ripple oscillations is well demonstrated (25, 26), it has been tacitly assumed that the ripple-associated, sequentially ordered firing of CA1 neurons is synaptically driven by the upstream CA3 cell assemblies (12, 15), largely because excitatory recurrent collaterals in the CA1 region are sparse (27). However, sequential activity may also emerge by local mechanisms, patterned by the different biophysical properties of CA1 pyramidal cells and their interactions with local interneurons, which discharge at different times during a ripple (28–30). A putative function of the rich variety of interneurons is temporal organization of principal cell spiking (29–32). We tested the “local-circuit” hypothesis by comparing the probability of participation and sequential firing of CA1 neurons during theta oscillations, natural spontaneous ripple events, and “synthetic” ripples induced by local optogenetic activation of pyramidal neurons.     

From the Cover: Neurons selective to the number of visual items in the corvid songbird endbrain

It is unknown whether anatomical specializations in the endbrains of different vertebrates determine the neuronal code to represent numerical quantity. Therefore, we recorded single-neuron activity from the endbrain of crows trained to judge the number of items in displays. Many neurons were tuned for numerosities irrespective of the physical appearance of the items, and their activity correlated with performance outcome. Comparison of both behavioral and neuronal representations of numerosity revealed that the data are best described by a logarithmically compressed scaling of numerical information, as postulated by the Weber–Fechner law. The behavioral and neuronal numerosity representations in the crow reflect surprisingly well those found in the primate association cortex. This finding suggests that distantly related vertebrates with independently developed endbrains adopted similar neuronal solutions to process quantity.Birds show elaborate quantification skills (1–3) that are of adaptive value in naturalistic situations like nest parasitism (4), food caching (5), or communication (6). The neuronal correlates of numerosity representations have only been explored in humans (7–9) and primates (10–18), and they have been found to reside in the prefrontal and posterior parietal neocortices. In contrast to primates, birds lack a six-layered neocortex. The birds’ lineage diverged from mammals 300 Mya (19), at a time when the neocortex had not yet developed from the pallium of the endbrain. Instead, birds developed different pallial parts as dominant endbrain structures (20, 21) based on convergent evolution, with the nidopallium caudolaterale (NCL) as a high-level association area (22–26). Where and how numerosity is encoded in vertebrates lacking a neocortex is unknown. Here, we show that neurons in the telencephalic NCL of corvid songbirds respond to numerosity and show a specific code for numerical information.     

Memory formation and retrieval of neuronal silencing in the auditory cortex

Sensory stimuli not only activate specific populations of cortical neurons but can also silence other populations. However, it remains unclear whether neuronal silencing per se leads to memory formation and behavioral expression. Here we show that mice can report optogenetic inactivation of auditory neuron ensembles by exhibiting fear responses or seeking a reward. Mice receiving pairings of footshock and silencing of a neuronal ensemble exhibited a fear response selectively to the subsequent silencing of the same ensemble. The valence of the neuronal silencing was preserved for at least 30 d and was susceptible to extinction training. When we silenced an ensemble in one side of auditory cortex for conditioning, silencing of an ensemble in another side induced no fear response. We also found that mice can find a reward based on the presence or absence of the silencing. Neuronal silencing was stored as working memory. Taken together, we propose that neuronal silencing without explicit activation in the cerebral cortex is enough to elicit a cognitive behavior.Cortical neurons exhibit spontaneous activity without explicit external stimuli (1–3), which may not only increase, but also be suppressed, by sensory stimuli (4, 5). For example, auditory stimuli suppress a subset of auditory cortical neurons in a frequency-dependent manner (5). Synaptic inhibition in the cerebral cortex is fundamental for neuronal modulation (6), including gain control (7), response selectivity (8, 9), and synchronized activities (10, 11). Inhibition-based modulations may contribute to stimulus-driven behaviors and associative memories of sensory stimuli (12); however, it remains unclear whether neuronal silencing (i.e., a transient reduction in firing rates from their spontaneous level) by itself can serve as a memory trace and bring about behavioral expressions. In this study, we tested this possibility by optogenetically silencing auditory cortical neurons.     

Connexin hemichannels contribute to spontaneous electrical activity in the human fetal cortex

Before the human cortex is able to process sensory information, young postmitotic neurons must maintain occasional bursts of action-potential firing to attract and keep synaptic contacts, to drive gene expression, and to transition to mature membrane properties. Before birth, human subplate (SP) neurons are spontaneously active, displaying bursts of electrical activity (plateau depolarizations with action potentials). Using whole-cell recordings in acute cortical slices, we investigated the source of this early activity. The spontaneous depolarizations in human SP neurons at midgestation (17–23 gestational weeks) were not completely eliminated by tetrodotoxin—a drug that blocks action potential firing and network activity—or by antagonists of glutamatergic, GABAergic, or glycinergic synaptic transmission. We then turned our focus away from standard chemical synapses to connexin-based gap junctions and hemichannels. PCR and immunohistochemical analysis identified the presence of connexins (Cx26/Cx32/Cx36) in the human fetal cortex. However, the connexin-positive cells were not found in clusters but, rather, were dispersed in the SP zone. Also, gap junction-permeable dyes did not diffuse to neighboring cells, suggesting that SP neurons were not strongly coupled to other cells at this age. Application of the gap junction and hemichannel inhibitors octanol, flufenamic acid, and carbenoxolone significantly blocked spontaneous activity. The putative hemichannel antagonist lanthanum alone was a potent inhibitor of the spontaneous activity. Together, these data suggest that connexin hemichannels contribute to spontaneous depolarizations in the human fetal cortex during the second trimester of gestation.In the adult brain, neuronal network activity is essentially driven by chemical synapses (1–3), whereas in the developing brain, neuronal activity is largely independent of sensory inputs (4–6). Membrane depolarizations during the earliest stages of brain development play an important role in the transition between the immature and mature signaling properties of neurons, as well as in shaping the mature functional neuronal network (7–12). Recent studies performed in the rodent model of cortical development have implicated subplate (SP) neurons as key regulators of early electrical activity and network oscillations (13, 14). Their essential role in the establishment of thalamocortical connections and cortical columns (15–18), extensive connectivity within the early synaptic network (19–21), and dense gap junction coupling (22, 23), as well as the abundant innervation by neuromodulatory transmitter systems (24, 25), put SP neurons in an ideal position to synchronize cortical activity during early development. Disruptions to the SP zone during development have been implicated in several major neurological disorders including schizophrenia, cerebral palsy, and autism (26, 27).Most of the physiological studies on SP neurons have been performed in the first postnatal week of rodent development [postnatal day 0 (P0) to P4], a time that corresponds more closely to the last (third) trimester of human gestation (28). Little is known about human SP neuron physiology in the first two trimesters, when massive proliferation, neuron migration and the initial stage of network formation are occurring in the human cerebral cortex (29, 30). General principles arising from the rodent model of cortical development should be tested in human neurons whenever possible, because it is well established in neurobiology that similar activity patterns can be produced by different sets of underlying conductances (31).Because of ethical and technical limitations associated with experimentation on human materials, there is a profound lack of information about physical, chemical, and biological agents that affect spontaneous electrical activity in human fetal cortex. Using acute brain slices obtained from postmortem human fetal tissue [17–23 gestational weeks (gw)], we analyzed the physiological effects of channel and receptor antagonists on SP neuron activity. We found that spontaneous bioelectric activity of human SP neurons is moderately influenced by drugs that block neuronal network activity and synaptic transmission and more strongly by drugs that interfere with the activity of connexin (Cx) pores. Several lines of our experimental data point to Cx hemichannels as significant contributors to spontaneous membrane depolarizations in human fetal cortex during the second trimester of gestation.     

Coherent delta-band oscillations between cortical areas correlate with decision making

Coherent oscillations in the theta-to-gamma frequency range have been proposed as a mechanism that coordinates neural activity in large-scale cortical networks in sensory, motor, and cognitive tasks. Whether this mechanism also involves coherent oscillations at delta frequencies (1–4 Hz) is not known. Rather, delta oscillations have been associated with slow-wave sleep. Here, we show coherent oscillations in the delta frequency band between parietal and frontal cortices during the decision-making component of a somatosensory discrimination task. Importantly, the magnitude of this delta-band coherence is modulated by the different decision alternatives. Furthermore, during control conditions not requiring decision making, delta-band coherences are typically much reduced. Our work indicates an important role for synchronous activity in the delta frequency band when large-scale, distant cortical networks coordinate their neural activity during decision making.Studies of the neural correlates of decision making in behaving monkeys have mainly been based on the analysis of firing rate patterns of neurons in individual cortical circuits, recorded one by one in succession, while trained monkeys perform sensory, motor, and cognitive tasks (1–3). These studies showed that the neuronal activities distributed across parietal and frontal lobe cortices correlate with processes that lead to decision making (4–7). However, how these spatially distant, cortical circuits coordinate their activities into a unified functional network during decision making remains poorly understood.It has been proposed that coherent oscillations of neuronal activities constitute a putative dynamical mechanism for mediating the interaction between different subsets of brain areas (8–11). Simultaneous recording from multiple intracortical areas in monkeys showed that the coherent higher frequency (beta and gamma bands) oscillations are linked to a broad variety of cognitive functions (12–18). Cortical oscillations at lower frequencies (theta and alpha bands) have also been discussed in terms of long-range integrative processes (19, 20). In fact, recent evidence showed theta-band coupling between visual area V4 and prefrontal cortex during short-term memory (21) and among the rat prefrontal cortex, ventral tegmental area, and hippocampus during working memory (22). It remains, however, probing whether coherent delta-band oscillations play a functional role in the interaction between cortical circuits. Delta-band oscillations are typically associated with slow-wave sleep (SWS; ref. 23), but an important question is whether delta-band oscillations during SWS and waking states represent the same underlying phenomenon (24). Recent findings associated delta-band oscillations in individual cortical areas with attention (25). In monkey primary visual cortex (26) and human motor cortex (27), delta-band oscillations entrain to the rhythm of external sensory events in an attention-dependent manner.Here, we examined whether coherent oscillations coordinate the activity of five simultaneously recorded cortical areas in the monkey performing a somatosensory discrimination task (7). We specifically focused on whether coherent delta-band oscillations play a significant functional role in linking cortical circuits during decision making.     

Brain–machine interface for eye movements

A number of studies in tetraplegic humans and healthy nonhuman primates (NHPs) have shown that neuronal activity from reach-related cortical areas can be used to predict reach intentions using brain–machine interfaces (BMIs) and therefore assist tetraplegic patients by controlling external devices (e.g., robotic limbs and computer cursors). However, to our knowledge, there have been no studies that have applied BMIs to eye movement areas to decode intended eye movements. In this study, we recorded the activity from populations of neurons from the lateral intraparietal area (LIP), a cortical node in the NHP saccade system. Eye movement plans were predicted in real time using Bayesian inference from small ensembles of LIP neurons without the animal making an eye movement. Learning, defined as an increase in the prediction accuracy, occurred at the level of neuronal ensembles, particularly for difficult predictions. Population learning had two components: an update of the parameters of the BMI based on its history and a change in the responses of individual neurons. These results provide strong evidence that the responses of neuronal ensembles can be shaped with respect to a cost function, here the prediction accuracy of the BMI. Furthermore, eye movement plans could be decoded without the animals emitting any actual eye movements and could be used to control the position of a cursor on a computer screen. These findings show that BMIs for eye movements are promising aids for assisting paralyzed patients.Brain–machine interfaces (BMIs) have been successfully used to predict reaches and arm movements (1–7). However, little effort has been concentrated on building a BMI based on eye movements. This gap is surprising because the motor and neuronal mechanisms of eye movements are very well understood and arguably simpler than those of arm movements. Specifically, eye movements are rapid and ballistic. The lateral intraparietal cortex (LIP) is ideally suited to be the site for a BMI based on eye movements (8). LIP neurons are known to encode eye movement plans, among other signals such as eye position (9–16). We recently showed that eye movement plans can be accurately predicted from the responses of populations of LIP neurons using Bayesian inference (16). The aim of the present study was twofold. First, a BMI was used with small neuronal ensembles of LIP neurons to predict, in real time, eye movement plans without the animals actually making eye movements. Second, the BMI application induced learning-related changes in the saccade system. Learning can produce changes in reach areas, but how learning-related changes occur at the level of LIP neuronal ensembles is still unclear (17, 18).Here, we show that the intended eye movement activity can be used to accurately position a cursor on a computer screen. These results suggest that an eye movement BMI can be used as a prosthetic to assist locked-in patients who cannot produce eye movements. Moreover, such an eye movement BMI can also be used to assist tetraplegic persons to decode intended limb movements by providing an extra channel of target position information (19). Learning, defined as an increase in the prediction accuracy, occurred at the level of neuronal ensembles, particularly for difficult predictions. The population learning had two components: an update of the parameters of the BMI based on its history and a change in the responses of individual neurons. These results provide strong evidence that the responses of neuronal ensembles can be shaped with respect to a cost function, which here is the prediction accuracy of the BMI. Such learning adds additional support for the utility of an eye movement BMI based on LIP activity.     

Optogenetic neuronal stimulation promotes functional recovery after stroke

Clinical and research efforts have focused on promoting functional recovery after stroke. Brain stimulation strategies are particularly promising because they allow direct manipulation of the target area’s excitability. However, elucidating the cell type and mechanisms mediating recovery has been difficult because existing stimulation techniques nonspecifically target all cell types near the stimulated site. To circumvent these barriers, we used optogenetics to selectively activate neurons that express channelrhodopsin 2 and demonstrated that selective neuronal stimulations in the ipsilesional primary motor cortex (iM1) can promote functional recovery. Stroke mice that received repeated neuronal stimulations exhibited significant improvement in cerebral blood flow and the neurovascular coupling response, as well as increased expression of activity-dependent neurotrophins in the contralesional cortex, including brain-derived neurotrophic factor, nerve growth factor, and neurotrophin 3. Western analysis also indicated that stimulated mice exhibited a significant increase in the expression of a plasticity marker growth-associated protein 43. Moreover, iM1 neuronal stimulations promoted functional recovery, as stimulated stroke mice showed faster weight gain and performed significantly better in sensory-motor behavior tests. Interestingly, stimulations in normal nonstroke mice did not alter motor behavior or neurotrophin expression, suggesting that the prorecovery effect of selective neuronal stimulations is dependent on the poststroke environment. These results demonstrate that stimulation of neurons in the stroke hemisphere is sufficient to promote recovery.Stroke is a major acute neurological insult that disrupts brain function and causes neuron death. Functional recovery after stroke has been observed and is currently attributed to both brain remodeling and plasticity (1–4). Structural and functional remodeling of areas next to an infarct or remote regions can alter signaling within bilateral neuronal networks and thus contribute to functional recovery (3–7). Rewiring of neural connections is mediated by electrical activity, which can activate a number of plasticity mechanisms, including the release of activity-dependent neurotrophins such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) (8–10). Both BDNF and NGF have been shown to improve recovery by enhancing axonal and dendritic sprouting (10–12).Tremendous effort has been focused on promoting recovery after stroke, including pharmacological treatment, rehabilitation (e.g., constraint-induced therapy), stem cell transplantation, and brain stimulation (1, 4, 13). Brain stimulation is a promising area of research because it allows direct activation and manipulation of the target area’s excitability (14–16). The primary motor cortex (M1) is a commonly stimulated area as it directly innervates the corticospinal tract to initiate movement (1, 7). Although electrical stimulation and transcranial magnetic stimulation show promise in promoting recovery (17, 18), these techniques are limited by imprecision and indiscriminate activation or inhibition of all cell types near the stimulated site; thus, they can produce undesired effects such as psychiatric and motor/speech problems (19–21). In addition, it has been difficult to elucidate the cell type and mechanisms driving recovery, as multiple cell types such as neurons, astrocytes, and oligodendrocytes have been shown to contribute to remodeling and recovery processes after stroke (5, 22–27).To elucidate whether activation of neurons alone can promote recovery, we used optogenetics to selectively manipulate the excitability of specific cell groups with millisecond-scale temporal precision in a manner more similar to endogenous neuronal firing patterns (21, 28, 29). This technique uses light-activated microbial proteins such as Channelrhodopsin 2 (ChR2), which depolarizes neurons when illuminated with blue light, or Halorhodopsin (NpHR), which hyperpolarizes neurons (21, 28, 29). Optogenetic approaches have been used in rodents to probe neuronal circuits for several neurological/neurodegenerative diseases, including Parkinson disease (30) and epilepsy (31). Recent studies have also used optogenetics to map functional organization after stroke (32–35). The safety and efficacy of using optogenetics in nonhuman primates has also been characterized (29, 36).In this study, we used optogenetics to selectively stimulate neurons in layer V of the ipsilesional primary motor cortex (iM1) and examine the effects of repeated neuronal stimulations in normal and stroke mice. Sensory-motor behavior tests were used to evaluate functional recovery after stroke, and plasticity-associated mechanisms, such as cerebral blood flow (CBF)/neurovascular coupling responses and activity-dependent neurotrophin expression, were investigated.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

Visual stimuli recruit intrinsically generated cortical ensembles

The cortical microcircuit is built with recurrent excitatory connections, and it has long been suggested that the purpose of this design is to enable intrinsically driven reverberating activity. To understand the dynamics of neocortical intrinsic activity better, we performed two-photon calcium imaging of populations of neurons from the primary visual cortex of awake mice during visual stimulation and spontaneous activity. In both conditions, cortical activity is dominated by coactive groups of neurons, forming ensembles whose activation cannot be explained by the independent firing properties of their contributing neurons, considered in isolation. Moreover, individual neurons flexibly join multiple ensembles, vastly expanding the encoding potential of the circuit. Intriguingly, the same coactive ensembles can repeat spontaneously and in response to visual stimuli, indicating that stimulus-evoked responses arise from activating these intrinsic building blocks. Although the spatial properties of stimulus-driven and spontaneous ensembles are similar, spontaneous ensembles are active at random intervals, whereas visually evoked ensembles are time-locked to stimuli. We conclude that neuronal ensembles, built by the coactivation of flexible groups of neurons, are emergent functional units of cortical activity and propose that visual stimuli recruit intrinsically generated ensembles to represent visual attributes.There is a growing consensus in neuroscience that ensembles of neurons working in concert, as opposed to single neurons, are the underpinnings of cognition and behavior (1–3). At the microcircuit level, the cortex is dominated by recurrent excitatory connections (4, 5). Such densely interconnected excitatory networks are ideal for generating reverberating activity (1, 6, 7) that could link neurons into functional neuronal ensembles. Moreover, most cortical neurons are part of highly distributed synaptic circuits, receiving inputs from and projecting outputs to, thousands of other neurons (8, 9). In fact, the basic excitatory neurons of the cortex, pyramidal cells, appear to be biophysically designed to perform large-scale integration of inputs (10). All of these structural features indicate the possibility that rather than relying on the firing of individual neurons, cortical circuits may generate responses built out of the coordinated activity of groups of neurons. These postulated emergent circuit states could represent the building blocks of mental and behavioral processes (1–3, 11).In the visual cortex, there has been continuing progress in understanding functional properties and receptive fields of single neurons using single-unit electrophysiology and optical imaging (12–14). These single-neuron studies have provided a solid foundation for neuroscience. However, the focus on single neurons may provide an incomplete picture of this highly distributed neural circuit (3, 15). In fact, in recent years, the network activity patterns of the primary visual cortex (V1) in vitro and in vivo have been shown to be highly structured in spatiotemporal properties (13, 16–18). For example, using voltage-sensitive imaging, one can measure large-scale cortical dynamics with high temporal resolution, albeit without single-cell resolution (19–22). At this bird’s-eye view, wave-like spontaneous spatiotemporal patterns of activity appear similar to those patterns measured during visual stimulation (19, 21, 22). These findings imply that groups of neurons are active together in the absence of any visual input and that the same groups of neurons are also active together in response to visual stimulation. However, to test this hypothesis, one must measure the circuit activity with single-cell resolution.With calcium imaging, multineuronal activity can be visualized with single-cell resolution (16, 23), so it has become possible to discern exactly which neurons are activated under spontaneous and visually evoked conditions, cell by cell. Indeed, calcium imaging of brain slices from mouse visual cortex has revealed that groups of neurons become coactive spontaneously (24, 25) and that the same groups of neurons can be triggered by stimulation of thalamic afferents (26, 27). However, the patterns of activity found in slices may differ from the patterns of activity in vivo. Therefore, to determine the relation between spontaneous and evoked cortical activity patterns properly, it is necessary to measure them in vivo.Using two-photon calcium imaging in vivo, we have now mapped the spontaneous reverberating activity patterns in the V1 from awake mice with single-cell resolution and analyzed their relation to the activity patterns evoked by visual stimulation. We find patterns of coactive neurons that we term “ensembles,” defined as “a group of items viewed as a whole rather than individually” (28). Although the mere existence of these coactive neurons does not prove their functional importance, we provide converging lines of evidence that these ensembles are, in fact, functional units of cortical activity. This work provides a step in the progression of defining neuronal ensembles, rather than receptive fields of individual cells, as a building block of cortical microcircuits and suggests that these intrinsic neuronal ensembles are recruited when the cortex performs some of its most basic functions.     

Natural asynchronies in audiovisual communication signals regulate neuronal multisensory interactions in voice-sensitive cortex

When social animals communicate, the onset of informative content in one modality varies considerably relative to the other, such as when visual orofacial movements precede a vocalization. These naturally occurring asynchronies do not disrupt intelligibility or perceptual coherence. However, they occur on time scales where they likely affect integrative neuronal activity in ways that have remained unclear, especially for hierarchically downstream regions in which neurons exhibit temporally imprecise but highly selective responses to communication signals. To address this, we exploited naturally occurring face- and voice-onset asynchronies in primate vocalizations. Using these as stimuli we recorded cortical oscillations and neuronal spiking responses from functional MRI (fMRI)-localized voice-sensitive cortex in the anterior temporal lobe of macaques. We show that the onset of the visual face stimulus resets the phase of low-frequency oscillations, and that the face–voice asynchrony affects the prominence of two key types of neuronal multisensory responses: enhancement or suppression. Our findings show a three-way association between temporal delays in audiovisual communication signals, phase-resetting of ongoing oscillations, and the sign of multisensory responses. The results reveal how natural onset asynchronies in cross-sensory inputs regulate network oscillations and neuronal excitability in the voice-sensitive cortex of macaques, a suggested animal model for human voice areas. These findings also advance predictions on the impact of multisensory input on neuronal processes in face areas and other brain regions.How the brain parses multisensory input despite the variable and often large differences in the onset of sensory signals across different modalities remains unclear. We can maintain a coherent multisensory percept across a considerable range of spatial and temporal discrepancies (1–4): For example, auditory and visual speech signals can be perceived as belonging to the same multisensory “object” over temporal windows of hundreds of milliseconds (5–7). However, such misalignment can drastically affect neuronal responses in ways that may also differ between brain regions (8–10). We asked how natural asynchronies in the onset of face/voice content in communication signals would affect voice-sensitive cortex, a region in the ventral “object” pathway (11) where neurons (i) are selective for auditory features in communication sounds (12–14), (ii) are influenced by visual “face” content (12), and (iii) display relatively slow and temporally variable responses in comparison with neurons in primary auditory cortical or subcortical structures (14–16).Neurophysiological studies in human and nonhuman animals have provided considerable insights into the role of cortical oscillations during multisensory conditions and for parsing speech. Cortical oscillations entrain to the slow temporal dynamics of natural sounds (17–20) and are thought to reflect the excitability of local networks to sensory inputs (21–24). Moreover, at least in auditory cortex, the onset of sensory input from the nondominant modality can reset the phase of ongoing auditory cortical oscillations (8, 25, 26), modulating the processing of subsequent acoustic input (8, 18, 22, 26–28). Thus, the question arises as to whether and how the phase of cortical oscillations in voice-sensitive cortex is affected by visual input.There is limited evidence on how asynchronies in multisensory stimuli affect cortical oscillations or neuronal multisensory interactions. Moreover, as we consider in the following, there are some discrepancies in findings between studies, leaving unclear what predictions can be made for regions beyond the first few stages of auditory cortical processing. In general there are two types of multisensory response modulations: Neuronal firing rates can be either suppressed or enhanced in multisensory compared with unisensory conditions (9, 12, 25, 29, 30). In the context of audiovisual communication Ghazanfar et al. (9) showed that these two types of multisensory influences are not fixed. Rather, they reported that the proportion of suppressed and enhanced multisensory responses in auditory cortical local-field potentials varies depending on the natural temporal asynchrony between the onset of visual (face) and auditory (voice) information. They interpret their results as an approximately linear change from enhanced to suppressed responses with increasing asynchrony between face movements and vocalization onset. In contrast, Lakatos et al. (8) found a cyclic, rather than linear, pattern of multisensory enhancement and suppression in auditory cortical neuronal responses as a function of increasing auditory–somatosensory stimulus onset asynchrony. This latter result suggests that the proportion of suppressed/enhanced multisensory responses varies nonlinearly (i.e., cyclically) with the relative onset timing of cross-modal stimuli. Although such results highlight the importance of multisensory asynchronies in regulating neural excitability, the differences between the studies prohibit generalizing predictions to other brain areas and thus leave the general principles unclear.In this study we aimed to address how naturally occurring temporal asynchronies in primate audiovisual communication signals affect both cortical oscillations and neuronal spiking activity in a voice-sensitive region. Using a set of human and monkey dynamic faces and vocalizations exhibiting a broad range of audiovisual onset asynchronies (Fig. 1), we demonstrate a three-way association between face–voice onset asynchrony, cross-modal phase resetting of cortical oscillations, and a cyclic pattern of dynamically changing proportions of suppressed and enhanced neuronal multisensory responses.Open in a separate windowFig. 1.Audiovisual primate vocalizations and visual–auditory delays. (A–C) Examples of audiovisual rhesus macaque coo (A and B) and grunt (C) vocalizations used for stimulation and their respective VA delays (time interval between the onset of mouth movement and the onset of the vocalization; red bars). The video starts at the onset of mouth movement, with the first frame showing a neutral facial expression, followed by mouth movements associated with the vocalization. Gray lines indicate the temporal position of the representative video frames (top row). The amplitude waveforms (middle row) and the spectrograms (bottom row) of the corresponding auditory component of the vocalization are displayed below.     

Rehabilitation drives enhancement of neuronal structure in functionally relevant neuronal subsets

We determined whether rehabilitation after cortical injury also drives dynamic dendritic and spine changes in functionally distinct subsets of neurons, resulting in functional recovery. Moreover, given known requirements for cholinergic systems in mediating complex forms of cortical plasticity, including skilled motor learning, we hypothesized that cholinergic systems are essential mediators of neuronal structural and functional plasticity associated with motor rehabilitation. Adult rats learned a skilled forelimb grasping task and then, underwent destructive lesions of the caudal forelimb region of the motor cortex, resulting in nearly complete loss of grasping ability. Subsequent intensive rehabilitation significantly enhanced both dendritic architecture and spine number in the adjoining rostral forelimb area compared with that in the lesioned animals that were not rehabilitated. Cholinergic ablation markedly attenuated rehabilitation-induced recovery in both neuronal structure and motor function. Thus, rehabilitation focused on an affected limb robustly drives structural compensation in perilesion cortex, enabling functional recovery.Studies over the past decade have indicated that the adult brain is structurally dynamic (1–3). Indeed, dendritic spines dynamically turn over in the adult brain (3, 4), and learning of novel tasks is associated with further increases in spine turnover (4). Moreover, total and stable increases in spine number together with enhanced dendritic complexity can be detected when analyses are focused specifically on neuronal subpopulations that are functionally related to a newly learned motor skill (5). For example, we recently reported that cortical layer V pyramidal neurons, which project to spinal segment C8 and are specifically engaged when learning a skilled forelimb grasping task, elaborate a 22% increase in apical dendritic spines and exhibit significant increases in dendritic branching and total dendritic length (5); an adjoining control population of cortical layer V pyramidal neurons that project to C4, which are not specifically shaped by the skilled motor task, exhibits no change in spines or dendritic complexity when the same task is learned (5). The detection of stable structural increases in neurons engaged by skilled motor learning in contrast to a lack of change in adjacent neurons that are not engaged by learning advances our understanding of mechanisms underlying experience-dependent cortical plasticity.Damage to the adult CNS also generates adaptive brain plasticity. For example, focal cortical lesions evoke cortical map plasticity (6, 7), extension of new axonal connections (7, 8), and neurogenesis (9). A very important and unresolved question in the neural plasticity and injury fields is whether rehabilitation—that is, specific retraining of injured neural circuits—can drive, alter, or enhance neural plasticity subsequent to brain lesions. Whereas extensive literature has shown that rehabilitation can increase the numbers of dendritic spines and dendritic complexity in the cortical hemisphere opposite a brain lesion (10–13) and is associated with improved skill in the limb unaffected by the lesion, effects of rehabilitation on neuronal structure in perilesioned cortex have not been described. Indeed, some studies suggest either stability or early loss of dendritic structure in perilesion cortex (14–16). However, knowing whether rehabilitation can drive adaptive brain plasticity could be essential in improving outcomes of numerous CNS disorders acquired in adulthood, including stroke, traumatic brain injury, and spinal cord injury.Prior studies that have sought to interrogate neuronal structure after injury have been limited by their use of nonspecific cellular sampling methods, such as Golgi–Cox staining or EM; these approaches lack the ability to specifically sample structural changes in neurons associated with specific tasks that are practiced in rehabilitation. Sampling from subpopulations of neurons mediating specific behaviors, such as skilled grasping in the motor cortex, may yield far more sensitive measures of changes in dendritic structure and spine number as a function of rehabilitation, fundamentally advancing our understanding of the role of experience and rehabilitation on structural neuronal plasticity.Another consideration in understanding cortical mechanisms underlying plasticity after CNS injury is the contribution of subcortical systems that modulate cortical activity, including cholinergic inputs. Studies have identified an essential role for cholinergic activation in modulating cortical plasticity associated with learning (17–19) and motor map plasticity that is evoked after lesions of the caudal forelimb region of the motor cortex (6, 20). These observations raise the possibility that cholinergic inputs to the motor cortex are also essential for generating neuronal structural adaptations in response to rehabilitation training after injury.In this study, we hypothesized that rehabilitation after injury to the adult brain drives adaptive plasticity, rebuilding spines and enhancing dendritic architecture in neurons surrounding the lesion site. We further hypothesize that these changes are cholinergic-dependent. We examined specific subpopulations of layer V cortical neurons directly related to the learning, loss, and subsequent relearning of skilled forelimb grasping, allowing detailed and specific sampling of structural parameters among subpopulations of neurons specifically engaged in the skilled grasping task.     

Emergence of functional subnetworks in layer 2/3 cortex induced by sequential spikes in vivo

During cortical circuit development in the mammalian brain, groups of excitatory neurons that receive similar sensory information form microcircuits. However, cellular mechanisms underlying cortical microcircuit development remain poorly understood. Here we implemented combined two-photon imaging and photolysis in vivo to monitor and manipulate neuronal activities to study the processes underlying activity-dependent circuit changes. We found that repeated triggering of spike trains in a randomly chosen group of layer 2/3 pyramidal neurons in the somatosensory cortex triggered long-term plasticity of circuits (LTPc), resulting in the increased probability that the selected neurons would fire when action potentials of individual neurons in the group were evoked. Significant firing pattern changes were observed more frequently in the selected group of neurons than in neighboring control neurons, and the induction was dependent on the time interval between spikes, N-methyl-D-aspartate (NMDA) receptor activation, and Calcium/calmodulin-dependent protein kinase II (CaMKII) activation. In addition, LTPc was associated with an increase of activity from a portion of neighboring neurons with different probabilities. Thus, our results demonstrate that the formation of functional microcircuits requires broad network changes and that its directionality is nonrandom, which may be a general feature of cortical circuit assembly in the mammalian cortex.Layer 2/3 neurons in the barrel cortex play a central role in integrative cortical processing (1–4). Neurons in layer 2/3 are interconnected with each other, and their axons and dendrites traverse adjacent barrel areas (5, 6). Recent calcium (Ca²⁺) imaging studies in awake animals showed that two very closely localized layer 2/3 pyramidal neurons are independently activated by different whiskers (7). In addition, adjacent layer 2/3 neurons have different receptive field properties; signals from different whiskers may emerge on different spines in the same neurons (8, 9). These findings suggest that the organization of functional subnetworks in somatosensory layer 2/3 is heterogeneous at the single-cell level and that microcircuits are assembled at a very fine scale (10). In vivo whole-cell recording experiments have also shown that most, but not all, layer 2/3 pyramidal neurons receive subthreshold depolarization by single-whisker stimulation with much broader receptive fields than neurons in layer 4 (11, 12). These anatomical and functional data suggest that electric signals relayed to the cortex by whisker activation are greatly intermingled within layer 2/3 neurons, and that studying the mechanisms by which these layer 2/3 neurons make connections may be critical for understanding the cortical network organizing principles underlying somatosensation.A previous modeling study suggested that spike timing-dependent plasticity (STDP) can lead to the formation of functional cortical columns and activity-dependent reorganization of neural circuits (13–16). However, how spikes arising in multiple neurons in vivo influence their connectivity is poorly understood. In this study using two-photon glutamate photolysis, which allowed us to control neuronal activity in a spatially and temporally precise manner, we examined activity-dependent cellular mechanisms during network rearrangement generated by repetitive spike trains in a group of neurons. We found that repetitive spikes on a group of neurons induced the probability of the neurons firing together. This circuit plasticity required spiking at short intervals among neurons and is expressed by N-methyl-D-aspartate (NMDA) receptor- and Calcium/calmodulin-dependent protein kinase II (CaMKII)-dependent long-lasting connectivity changes. The probability of firing was differentially affected by the order of the spike sequence but was not dependent on the physical distance between neurons. Thus, our data show that neuronal connectivity within a functional subnetwork is established in not only a preferred but also a directional manner.     

Cortically projecting basal forebrain parvalbumin neurons regulate cortical gamma band oscillations

Cortical gamma band oscillations (GBO, 30–80 Hz, typically ∼40 Hz) are involved in higher cognitive functions such as feature binding, attention, and working memory. GBO abnormalities are a feature of several neuropsychiatric disorders associated with dysfunction of cortical fast-spiking interneurons containing the calcium-binding protein parvalbumin (PV). GBO vary according to the state of arousal, are modulated by attention, and are correlated with conscious awareness. However, the subcortical cell types underlying the state-dependent control of GBO are not well understood. Here we tested the role of one cell type in the wakefulness-promoting basal forebrain (BF) region, cortically projecting GABAergic neurons containing PV, whose virally transduced fibers we found apposed cortical PV interneurons involved in generating GBO. Optogenetic stimulation of BF PV neurons in mice preferentially increased cortical GBO power by entraining a cortical oscillator with a resonant frequency of ∼40 Hz, as revealed by analysis of both rhythmic and nonrhythmic BF PV stimulation. Selective saporin lesions of BF cholinergic neurons did not alter the enhancement of cortical GBO power induced by BF PV stimulation. Importantly, bilateral optogenetic inhibition of BF PV neurons decreased the power of the 40-Hz auditory steady-state response, a read-out of the ability of the cortex to generate GBO used in clinical studies. Our results are surprising and novel in indicating that this presumptively inhibitory BF PV input controls cortical GBO, likely by synchronizing the activity of cortical PV interneurons. BF PV neurons may represent a previously unidentified therapeutic target to treat disorders involving abnormal GBO, such as schizophrenia.Gamma band oscillations (GBO) (30–80 Hz) recorded in the cortical EEG have generated intense interest in recent years. Theoretical and experimental work suggests that GBO play a crucial role in feature binding (1), attention (2), and consciousness (3). Furthermore, GBO abnormalities have been reported in severe neuropsychiatric disorders, such as Alzheimer’s disease (4) and schizophrenia (5, 6). Animal studies have shown that GBO are generated by fast-spiking cortical interneurons containing the calcium-binding protein parvalbumin (PV) (7, 8), which postmortem clinical studies have shown to be abnormal in schizophrenia (9). Animal models of schizophrenia have shown that alterations in the function of cortical PV interneurons are strongly correlated with deficits in GBO and cognition (10, 11). Thus, understanding the regulation of cortical PV interneurons and GBO is essential for developing treatments for schizophrenia and other disorders in which GBO are abnormal.The ability of the cortex to generate GBO fluctuates according to the level of arousal and consciousness. GBO are reduced in non-rapid eye movement (NREM) sleep (12), deep anesthesia (13), and vegetative state (14). Thus, cortical GBO are regulated by the ascending reticular activating system (ARAS), originally described by Moruzzi and Magoun (15). Electrical stimulation of the origin of the ARAS in the brainstem reticular formation elicited cortical low-voltage fast activity (15) and enhanced GBO (16), suggesting that increased activity in the ARAS is responsible for behavioral state-dependent changes in cortical GBO.Lesion experiments in rodents suggested that cortically projecting neurons in the basal forebrain (BF), the final node of the ventral arm of the ARAS, may be particularly important in controlling cortical activation and GBO (17–19). Although these studies indicated the importance of the BF, they did not reveal which specific BF cell types are involved. Although selective BF cholinergic lesions cause a modest reduction in the amplitude of cortical fast oscillations, they do not abolish them (17), suggesting that additional neuronal subtypes are involved.Among the BF noncholinergic neurons, GABAergic neurons are of particular interest because a significant minority discharges at high rates (20–60 Hz) in association with cortical activation during wakefulness and REM sleep (20). Many cortically projecting BF GABAergic neurons contain the calcium-binding protein PV, and a large majority of BF PV neurons are GABAergic (21, 22). Recordings from identified BF PV neurons in anesthetized animals revealed they increase their discharge rate during cortical activation induced by sensory stimulation (23). Furthermore, reduced cortical activation following BF lesions was correlated with the extent of PV neuronal loss (17). Interestingly, BF GABAergic projections to cortical GABAergic interneurons containing PV (24, 25) have been demonstrated, although direct BF PV→cortical PV connections remain to be shown. When considered together, the physiology and anatomical connections of BF GABAergic/PV neurons suggest that they are ideally positioned to control cortical GBO (Fig. 1A). However, this hypothesis has not been tested directly. Thus, here we tested the effect of selective excitation of BF PV neurons on the cortical EEG and the effect of selective inhibition on the 40-Hz auditory steady-state response (ASSR), a test of the ability of the cortex to generate GBO (26) widely used in clinical studies of anesthesia (27), hearing loss (28), and schizophrenia (5, 29).Fig. 1.BF PV neurons control cortical GBO, typically at ∼40 Hz. (A) The experimental model tested in this study. Cortical GBO are generated through the interaction of cortical GABAergic PV interneurons and pyramidal neurons (PYR). BF PV projection neurons ...     

Assessing the sensitivity of diffusion MRI to detect neuronal activity directly

Functional MRI (fMRI) is widely used to study brain function in the neurosciences. Unfortunately, conventional fMRI only indirectly assesses neuronal activity via hemodynamic coupling. Diffusion fMRI was proposed as a more direct and accurate fMRI method to detect neuronal activity, yet confirmative findings have proven difficult to obtain. Given that the underlying relation between tissue water diffusion changes and neuronal activity remains unclear, the rationale for using diffusion MRI to monitor neuronal activity has yet to be clearly established. Here, we studied the correlation between water diffusion and neuronal activity in vitro by simultaneous calcium fluorescence imaging and diffusion MR acquisition. We used organotypic cortical cultures from rat brains as a biological model system, in which spontaneous neuronal activity robustly emerges free of hemodynamic and other artifacts. Simultaneous fluorescent calcium images of neuronal activity are then directly correlated with diffusion MR signals now free of confounds typically encountered in vivo. Although a simultaneous increase of diffusion-weighted MR signals was observed together with the prolonged depolarization of neurons induced by pharmacological manipulations (in which cell swelling was demonstrated to play an important role), no evidence was found that diffusion MR signals directly correlate with normal spontaneous neuronal activity. These results suggest that, whereas current diffusion MR methods could monitor pathological conditions such as hyperexcitability, e.g., those seen in epilepsy, they do not appear to be sensitive or specific enough to detect or follow normal neuronal activity.Developing a direct MRI method to detect neuronal activity in vivo and noninvasively is a major focus in neuroscience. Progress in this area is required to improve our understanding of normal brain function, and in a clinical setting, to develop new tools for studying normal and abnormal development to diagnose diseases and disorders of the brain. Functional MRI (fMRI) has been widely used in the cognitive neurosciences since its invention in the 1990s (1–3). The most widely used fMRI method, blood-oxygenation-level-dependent (BOLD) MRI, detects hemodynamic changes in the brain, which only indirectly reflects neuronal activity. Moreover, its hemodynamic origin limits both its spatial and temporal resolution and its interpretation as a direct proxy for neuronal activity (4, 5).More recently, several MRI methods were proposed to provide more direct measures of neuronal excitation (6). In particular, diffusion MRI, a method to measure the apparent diffusivity of water within tissues (7–9), has been suggested as a direct functional imaging method to detect neuronal activity (10–13). Early in vivo experiments in both humans and animals reported small but significant increases in highly diffusion-weighted MRI signals, which were ascribed to changes directly induced by the underlying neuronal activity rather than indirect hemodynamic changes (10–13). In vitro experiments on brain slices (14, 15) and spinal cord (16) reported similar reductions in water diffusivity under conditions of extreme hyperexcitability using strong pharmacologic stimulants.However, functional diffusion MRI (fDMRI) has not been widely used or adopted since its introduction almost two decades ago. Two major reasons for this may be a dearth of experiments that convincingly establish its neurophysiological basis and the poor reproducibility of the originally reported changes in diffusion MRI signals by different laboratories. The inability to detect the predicted changes using fDMRI and the possible confounds of hemodynamic contributions in fDMRI measurements in vivo do not argue for a robust connection between changes in diffusion MRI and underlying neuronal activity (17–20). Thus, “ground-truth” experiments, potentially establishing a connection between the changes in diffusion MRI and underlying neuronal activity, are needed, particularly to shed light on the possible biophysical basis of the fDMRI signal.Recently, we developed a novel test bed that could be used to assess non–hemodynamic-based functional MRI methods, in which MR signal acquisition and intracellular calcium fluorescence imaging to monitor neuronal activity can be performed simultaneously on organotypic cortical cultures from rat brains (21). The organotypic cortex culture is a well-characterized biological model of neuronal activity free of hemodynamic, respiratory, and other physiological confounds. Not only is the in vivo cortical cytoarchitecture preserved (including cortical layers and cortical cell types), but neuronal activity in the culture also displays bursts of spontaneous neuronal avalanches grouped into so-called up-states and separated by periods of low activity (22–25), resembling resting neuronal activity in vivo (26–28). Specifically, fluorometric calcium (Ca²⁺) imaging is used to detect intracellular Ca²⁺ concentration changes that closely follow action potentials in neurons under normal conditions and provide a direct method for detecting neuronal spiking activity in a neuronal network (29, 30). This test bed thus allows one to study precisely and accurately temporal correlations between the candidate functional MR signals, which are free of the usual in vivo confounds, and the underlying neuronal spiking activity by using an independent intracellular Ca²⁺ imaging experiment (21).In the current study, diffusion MR signals are obtained simultaneously with intracellular calcium fluorescence imaging of the organotypic cortex culture. The direct effects of neuronal activity on the diffusion MR signals are studied by time-series analysis of the simultaneous calcium and MR signals during normal neuronal activity and in different pathological states, which include induced hyperexcitability by kainic acid (kainate) and potassium, disinhibition by picrotoxin (PTX), suppression of excitability by tetrodotoxin (TTX), and cell volume modulation caused by osmotic pressure challenges. On the basis of these findings, it is possible to assess the prospect of detecting normal and abnormal neuronal activity using fDMRI and to better understand the relationship between fDMRI changes and biophysical mechanisms associated with neuronal excitation.