Sergei A. Kirov

Dendrites are more spiny on mature hippocampal neurons when synapses are inactivated

Dendrites of CA1 pyramidal neurons in mature rat hippocampal slices were exposed to different levels of synaptic activation. In some slices, synaptic transmission was blocked with glutamate receptor antagonists, sodium and calcium channel blockers and/or a nominally calcium-free medium with high magnesium. In other slices, synapses were activated with low-frequency control stimulation or repeated tetanic stimulation. In slices with blocked synaptic transmission, dendrites were spinier than in either of the activated states. Thus, mature neurons can increase their numbers of spines, possibly compensating for lost synaptic activity.

Plasticity of perisynaptic astroglia during synaptogenesis in the mature rat hippocampus.

Astroglia are integral components of synapse formation and maturation during development. Less is known about how astroglia might influence synaptogenesis in the mature brain. Preparation of mature hippocampal slices results in synapse loss followed by recuperative synaptogenesis during subsequent maintenance in vitro. Hence, this model system was used to discern whether perisynaptic astroglial processes are similarly plastic, associating more or less with recently formed synapses in mature brain slices. Perisynaptic astroglia was quantified through serial section electron microscopy in perfusion‐fixed or sliced hippocampus from adult male Long‐Evans rats that were 65–75 days old. Fewer synapses had perisynaptic astroglia in the recovered hippocampal slices (42.4% ± 3.4%) than in the intact hippocampus (62.2% ± 2.6%), yet synapses were larger when perisynaptic astroglia was present (0.055 ± 0.003 μm2) than when it was absent (0.036 ± 0.004 μm2) in both conditions. Importantly, the length of the synaptic perimeter surrounded by perisynaptic astroglia and the distance between neighboring synapses was not proportional to synapse size. Instead, larger synapses had longer astroglia‐free perimeters where substances could escape from or enter into the synaptic clefts. Thus, smaller presumably newer synapses as well as established larger synapses have equal access to extracellular glutamate and secreted astroglial factors, which may facilitate recuperative synaptogenesis. These findings suggest that as synapses enlarge and release more neurotransmitter, they attract astroglial processes to a discrete portion of their perimeters, further enhancing synaptic efficacy without limiting the potential for cross talk with neighboring synapses in the mature rat hippocampus.

Dendritic spines disappear with chilling but proliferate excessively upon rewarming of mature hippocampus

More dendritic spine synapses occur on mature neurons in hippocampal slices by 2 h of incubation in vitro, than in perfusion-fixed hippocampus. What conditions initiate this spinogenesis and how rapidly do the spines begin to proliferate on mature neurons? To address these questions, CA1 field of the hippocampus neurons expressing green fluorescent protein in living slices from mature mice were imaged with two-photon microscopy. Spines disappeared and dendrites were varicose immediately after slice preparation in ice-cold artificial cerebrospinal fluid (ACSF). Electron microscopy (EM) revealed disrupted dendritic cytoplasm, enlarged or free-floating postsynaptic densities, and excessive axonal endocytosis. Upon warming dendritic varicosities shrank and spines rapidly reappeared within a few minutes illustrating the remarkable resilience of mature hippocampal neurons in slices. When membrane impermeant sucrose was substituted for NaCl in ACSF dendrites remained spiny at ice-cold temperatures and EM revealed less disruption. Nevertheless, spine number and length increased within 30 min in warm ACSF even when the extracellular calcium concentration was zero and synaptic transmission was blocked. When slices were first recovered for several hours and then chilled in 6 degrees C ACSF many spines disappeared and the dendrites became varicose. Upon re-warming varicosities shrank and spines reemerged in the same position from which they disappeared. In addition, new spines formed and spines were longer suggesting that chilling, not the initial injury from slicing, caused the spines to disappear while re-warming triggered the spine proliferation on mature neurons. The new spines might be a substrate for neuronal recovery of function, when neurons have been chilled or exposed to other traumatic conditions that disrupt ionic homeostasis.

Real-time passive volume responses of astrocytes to acute osmotic and ischemic stress in cortical slices and in vivo revealed by two-photon microscopy.

The brain swells over the several minutes that follow stroke onset or acute hypo‐osmotic stress because cells take up water. Measuring the volume responses of single neurons and glia has necessarily been confined to isolated or cultured cells. Two‐photon laser scanning microscopy enables real‐time visualization of cells functioning deep within living neocortex in vivo or in brain slices under physiologically relevant osmotic and ischemic stress. Astrocytes and their processes expressing green fluorescent protein in murine cortical slices swelled in response to 20 min of overhydration (−40 mOsm) and shrank during dehydration (+40 or +80 mOsm) at 32–34°C. Minute‐by‐minute monitoring revealed no detectable volume regulation during these osmotic challenges, particularly during the first 5 min. Astrocytes also rapidly swelled in response to elevated [K+]o for 3 min or oxygen/glucose deprivation (OGD) for 10 min. Post‐OGD, astroglial volume recovered quickly when slices were re‐supplied with oxygen and glucose, while neurons remained swollen with beaded dendrites. In vivo, rapid astroglial swelling was confirmed within 6 min following intraperitoneal water injection or during the 6–12 min following cardiac arrest. While the astrocytic processes were clearly swollen, the extent of the astroglial arbor remained unchanged. Thus, in contrast to osmo‐resistant pyramidal neurons (Andrew et al., 2007 ) that lack known aquaporins, astrocytes passively respond to acute osmotic stress, reflecting functional aquaporins in their plasma membrane. Unlike neurons, astrocytes better recover from brief ischemic insult in cortical slices, probably because their aquaporins facilitate water efflux.

Timing of neuronal and glial ultrastructure disruption during brain slice preparation and recovery in vitro

Hippocampal slices often have more synapses than perfusion‐fixed hippocampus, but the cause of this synaptogenesis is unclear. Ultrastructural evidence for synaptogenic triggers during slice preparation was investigated in 21‐day‐old rats. Slices chopped under warm or chilled conditions and fixed after 0, 5, 25, 60, or 180 minutes of incubation in an interface chamber were compared with hippocampi fixed by perfusion or by immersion of the whole hippocampus. There was no significant synaptogenesis in these slices compared with perfusion‐fixed hippocampus, but there were other structural changes during slice preparation and recovery in vitro. Whole hippocampus and slices prepared under warm conditions exhibited an increase in axonal coated vesicles, suggesting widespread neurotransmitter release. Glycogen granules were depleted from astrocytes and neurons in 0‐min slices, began to reappear by 1 hour, and had fully recovered by 3 hours. Dendritic microtubules were initially disassembled in slices, but reassembled into normal axial arrays after 5 minutes. Microtubules were short at 5 minutes (12.3 ± 1.1 μm) but had recovered normal lengths by 3 hours (84.6 ± 20.0 μm) compared with perfusion‐fixed hippocampus (91 ± 22 μm). Microtubules appeared transiently in 15 ± 3% and 9 ± 4% of dendritic spines 5 and 25 minutes after incubation, respectively. Spine microtubules were absent from perfusion‐fixed hippocampus and 3‐hour slices. Ice‐cold dissection and vibratomy in media that blocked activity initially produced less glycogen loss, coated vesicles, and microtubule disassembly. Submersing these slices in normal oxygenated media at 34°C led to glycogen depletion, as well as increased coated vesicles and microtubule disassembly within 1 minute. J. Comp. Neurol. 465:90–103, 2003.

Specific regulation of NRG1 isoform expression by neuronal activity

Neuregulin 1 (NRG1) is a trophic factor that has been implicated in neural development, neurotransmission, and synaptic plasticity. NRG1 has multiple isoforms that are generated by usage of different promoters and alternative splicing of a single gene. However, little is known about NRG1 isoform composition profile, whether it changes during development, or the underlying mechanisms. We found that each of the six types of NRG1 has a distinct expression pattern in the brain at different ages, resulting in a change in NRG1 isoform composition. In both human and rat, the most dominant are types III and II, followed by either type I or type V, while types IV and VI are the least abundant. The expression of NRG1 isoforms is higher in rat brains at ages of E13 and P5 (in particular type V), suggesting roles in early neural development and in the neonatal critical period. At the cellular level, the majority of NRG1 isoforms (types I, II, and III) are expressed in excitatory neurons, although they are also present in GABAergic neurons and astrocytes. Finally, the expression of each NRG1 isoform is distinctly regulated by neuronal activity, which causes significant increase in type I and IV NRG1 levels. Neuronal activity regulation of type IV expression requires a CRE cis-element in the 5′ untranslated region (UTR) that binds to CREB. These results indicate that expression of NRG1 isoforms is regulated by distinct mechanisms, which may contribute to versatile functions of NRG1 and pathologic mechanisms of brain disorders such as schizophrenia.

Recurrent Spontaneous Spreading Depolarizations Facilitate Acute Dendritic Injury in the Ischemic Penumbra

Spontaneous spreading depolarizations (SDs) occur in the penumbra surrounding ischemic core. These SDs, often referred to as peri-infarct depolarizations, cause vasoconstriction and recruitment of the penumbra into the ischemic core in the critical first hours after focal ischemic stroke; however, the real-time spatiotemporal dynamics of SD-induced injury to synaptic circuitry in the penumbra remain unknown. A modified cortical photothrombosis model was used to produce a square-shaped lesion surrounding a penumbra-like “area at risk” in middle cerebral artery territory of mouse somatosensory cortex. Lesioning resulted in recurrent spontaneous SDs. In vivo two-photon microscopy of green fluorescent protein-expressing neurons in this penumbra-like area at risk revealed that SDs were temporally correlated with rapid (<6 s) dendritic beading. Dendrites quickly (<3 min) recovered between SDs to near-control morphology until the occurrence of SD-induced terminal dendritic injury, signifying acute synaptic damage. SDs are characterized by a breakdown of ion homeostasis that can be recovered by ion pumps if the energy supply is adequate. Indeed, the likelihood of rapid dendritic recovery between SDs was correlated with the presence of nearby flowing blood vessels, but the presence of such vessels was not always sufficient for rapid dendritic recovery, suggesting that energy needs for recovery exceeded energy supply of compromised blood flow. We propose that metabolic stress resulting from recurring SDs facilitates acute injury at the level of dendrites and dendritic spines in metabolically compromised tissue, expediting penumbral recruitment into the ischemic core.

Synaptogenesis on mature hippocampal dendrites occurs via filopodia and immature spines during blocked synaptic transmission

During development, dendritic spines emerge as stubby protrusions from single synapses on dendritic shafts or from retracting filopodia, many of which have more than one synapse. These structures are rarely encountered in the mature brain. Recently, confocal and two‐photon microscopy have revealed a proliferation of new filopodia‐like protrusions in mature hippocampal slices, especially when synaptic transmission was blocked. It was not known whether these protrusions have synapses nor whether they are accompanied by the other immature spine forms. Here, reconstruction from serial section electron microscopy (ssEM) was used to answer these questions. Acute hippocampal slices from mature male rats, ages 56 and 63 days, were maintained in vitro in control medium or in a nominally calcium‐free medium with high magnesium, glutamate receptor antagonists, and sodium and calcium channel blockers. At the end of each 8‐hour experiment, all slices were fixed, coded, and processed for ssEM. In agreement with light microscopy, there were more filopodia along dendrites in slices with blocked synaptic transmission. These filopodia were identified by their pointy tips and either the absence of synapses or presence of multiple synapses along them. There was also a proliferation of stubby spines. Filopodia along mature dendrites were typically shorter than developmental filopodia, with outgrowth likely being constrained by reduced extracellular space and compact neuropil, providing numerous candidate presynaptic partners in the vicinity of the mature dendrites. These findings suggest that synaptogenesis and spine formation are readily initiated under conditions of reduced activity in the mature brain. J. Comp. Neurol. 484:183–190, 2005.

Age-dependence in the homeostatic upregulation of hippocampal dendritic spine number during blocked synaptic transmission

Homeostatic regulation of spine number in mature hippocampal neurons results in more dendritic spines when synaptic transmission is blocked, providing a mechanism to compensate for diminished synaptic input. It is unsettled whether blockade of synaptic transmission also elevates spine number during development. To address this question, synaptic transmission was blocked in rat hippocampal slices during critical developmental stages of spine formation at postnatal days (P) 6-P22 and compared to adults. CA1 pyramidal cells were labeled with DiI and maintained for 5 h in one of three conditions, control artificial cerebrospinal fluid (ACSF), block media containing synaptic transmission antagonists in ACSF, or block media containing synaptic transmission antagonists in a nominally calcium-free ACSF with high magnesium. Slices were fixed in mixed aldehydes, sectioned, and the lateral dendrites were imaged in stratum radiatum with confocal microscopy. Dendritic spine density was quantified per unit length of dendrite. At P6-7 there were only a few protrusions emerging from the dendrites, which were predominantly filopodia-like in appearance. At both P11-12 and P15-16 there was a mixture of dendritic spines and filopodia-like structures. By P20-22 dendritic spines predominated and spine density was about 82% of the adult level. Dendritic spine density increased during blocked synaptic transmission at P20-22 as in adults, but was unchanged during blockade at younger ages. When extracellular calcium was nominally zero, dendritic spine density further increased on P20-22 dendrites as in adults. In contrast, spine density decreased along P11-12 dendrites under the nominally zero calcium condition. Under control conditions, dendritic protrusions were longer at P6-7 than at all other ages, which did not differ from one another. When synaptic transmission was blocked, dendritic protrusions further elongated at P6-7 only. Under the nominally zero calcium condition with blocked synaptic transmission, dendritic protrusions shortened at P11-12 only. These findings reveal age-dependent changes in the manifestation of homeostatic control of dendritic spines that could be mediated by maturational changes in mechanisms regulating postsynaptic calcium.

The continuum of spreading depolarizations in acute cortical lesion development: Examining Leão's legacy.

A modern understanding of how cerebral cortical lesions develop after acute brain injury is based on Aristides Leão’s historic discoveries of spreading depression and asphyxial/anoxic depolarization. Treated as separate entities for decades, we now appreciate that these events define a continuum of spreading mass depolarizations, a concept that is central to understanding their pathologic effects. Within minutes of acute severe ischemia, the onset of persistent depolarization triggers the breakdown of ion homeostasis and development of cytotoxic edema. These persistent changes are diagnosed as diffusion restriction in magnetic resonance imaging and define the ischemic core. In delayed lesion growth, transient spreading depolarizations arise spontaneously in the ischemic penumbra and induce further persistent depolarization and excitotoxic damage, progressively expanding the ischemic core. The causal role of these waves in lesion development has been proven by real-time monitoring of electrophysiology, blood flow, and cytotoxic edema. The spreading depolarization continuum further applies to other models of acute cortical lesions, suggesting that it is a universal principle of cortical lesion development. These pathophysiologic concepts establish a working hypothesis for translation to human disease, where complex patterns of depolarizations are observed in acute brain injury and appear to mediate and signal ongoing secondary damage.