Osmotic Edema Rapidly Increases Neuronal Excitability Through Activation of NMDA Receptor-Dependent Slow Inward Currents in Juvenile and Adult Hippocampus.

Cellular edema (cell swelling) is a principal component of numerous brain disorders including ischemia, cortical spreading depression, hyponatremia, and epilepsy. Cellular edema increases seizure-like activity in vitro and in vivo, largely through nonsynaptic mechanisms attributable to reduction of the extracellular space. However, the types of excitability changes occurring in individual neurons during the acute phase of cell volume increase remain unclear. Using whole-cell patch clamp techniques, we report that one of the first effects of osmotic edema on excitability of CA1 pyramidal cells is the generation of slow inward currents (SICs), which initiate after approximately 1 min. Frequency of SICs increased as osmolarity decreased in a dose-dependent manner. Imaging of real-time volume changes in astrocytes revealed that neuronal SICs occurred while astrocytes were still in the process of swelling. SICs evoked by cell swelling were mainly nonsynaptic in origin and NMDA receptor-dependent. To better understand the relationship between SICs and changes in neuronal excitability, recordings were performed in increasingly physiological conditions. In the absence of any added pharmacological reagents or imposed voltage clamp, osmotic edema induced excitatory postsynaptic potentials and burst firing over the same timecourse as SICs. Like SICs, action potentials were blocked by NMDAR antagonists. Effects were more pronounced in adult (8-20 weeks old) compared with juvenile (P15-P21) mice. Together, our results indicate that cell swelling triggered by reduced osmolarity rapidly increases neuronal excitability through activation of NMDA receptors. Our findings have important implications for understanding nonsynaptic mechanisms of epilepsy in relation to cell swelling and reduction of the extracellular space

Motor Function and Dopamine Release Measurements in Transgenic Huntington’s Disease Model Rats

Huntington’s disease (HD) is a fatal, genetic, neurodegenerative disorder characterized by deficits in motor and cognitive function. Here, we have quantitatively characterized motor deficiencies and dopamine release dynamics in transgenic HD model rats. Behavioral analyses were conducted using a newly-developed force-sensing runway and a previously-developed force-plate actometer. Gait disturbances were readily observed in transgenic HD rats at 12 to 15 months of age. Additionally, dopamine system challenge by ip injection of amphetamine also revealed that these rats were resistant to the expression of focused stereotypy compared to wild-type controls. Moreover, dopamine release, evoked by the application of single and multiple electrical stimulus pulses applied at different frequencies, and measured using fast-scan cyclic voltammetry at carbon-fiber microelectrodes, was diminished in transgenic HD rats compared to age-matched wild-type control rats. Collectively, these results underscore the potential contribution of dopamine release alterations to the expression of motor impairments in transgenic HD rats

Hippocampal and Cortical Pyramidal Neurons Swell in Parallel with Astrocytes during Acute Hypoosmolar Stress

Normal nervous system function is critically dependent on the balance of water and ions in the extracellular space (ECS). Pathological reduction in brain interstitial osmolarity results in osmotically-driven flux of water into cells, causing cellular edema which reduces the ECS and increases neuronal excitability and risk of seizures. Astrocytes are widely considered to be particularly susceptible to cellular edema due to selective expression of the water channel aquaporin-4 (AQP4). The apparent resistance of pyramidal neurons to osmotic swelling has been attributed to lack of functional water channels. In this study we report rapid volume changes in CA1 pyramidal cells in hypoosmolar ACSF (hACSF) that are equivalent to volume changes in astrocytes across a variety of conditions. Astrocyte and neuronal swelling was significant within 1 min of exposure to 17 or 40% hACSF, was rapidly reversible upon return to normosmolar ACSF, and repeatable upon re-exposure to hACSF. Neuronal swelling was not an artifact of patch clamp, occurred deep in tissue, was similar at physiological vs. room temperature, and occurred in both juvenile and adult hippocampal slices. Neuronal swelling was neither inhibited by TTX, nor by antagonists of NMDA or AMPA receptors, suggesting that it was not occurring as a result of excitotoxicity. Surprisingly, genetic deletion of AQP4 did not inhibit, but rather augmented, astrocyte swelling in severe hypoosmolar conditions. Taken together, our results indicate that neurons are not osmoresistant as previously reported, and that osmotic swelling is driven by an AQP4-independent mechanism

Rapid Changes in Neuronal Excitability During Osmotic Edema in Juvenile and Adult Hippocampus Through NMDA Receptor Activation

Cerebral edema affects millions of people worldwide and is associated with a plethora of diseases, disorders, and conditions such as traumatic brain injury, stroke, cardiac arrest, autism and epilepsy. Cellular edema has been known to increase epileptiform activity and seizure susceptibility in vitro and in vivo. However, the identity of the cell types undergoing volume increases and the types of excitability changes that occur in neurons remain unclear. Electrophysiological whole-cell patch clamp techniques were used to record currents and potentials from CA1 pyramidal neurons during the application of hypoosmolar ACSF (hACSF) in acutely isolated hippocampal slices from mice. Hypoosmolar ACSF evoked slow inward currents (SICs) in neurons, which initiated after ~1 minute of hACSF application. Neuronal excitability increased as osmolarity decreased in a dose-dependent manner. Even 5% reductions in osmolarity were sufficient to significantly increase neuronal excitability. In addition, hACSF induced neuronal firing of action potentials (APs), independent of AMPA receptor activation. Neuronal excitability was also increased during application of hACSF while blocking both APs and AMPA receptors. Increased sub-threshold EPSPs, neuronal APs, and bursting activity were also evoked in the presence of Mg2+, suggesting that hypoosmolar insults increase neuronal excitability under more physiological conditions. Hypoosmolar insults increased neuronal excitability in both juvenile (P15-P21) and adult 2- to 5-month-old) mice. Bursting activity in adult mice during osmotic insult was elevated compared to juvenile mice. During hypoosmolar insults the frequency of SICs recorded at physiological temperature were significantly elevated from SICs recorded at room temperature. SICs were potentiated by D-serine, and blocked by both DL-AP5 and the NR2B specific compound Ro25-6981. Together, these results indicate that osmotic insults produce cellular edema in both neurons and astrocytes, and increase neuronal excitability within minutes through a combination of synaptic and non-synaptic activation of glutamate receptors

Rapid Changes in Neuronal Excitability During Osmotic Edema in Juvenile and Adult Hippocampus Through NMDA Receptor Activation

Cerebral edema affects millions of people worldwide and is associated with a plethora of diseases, disorders, and conditions such as traumatic brain injury, stroke, cardiac arrest, autism and epilepsy. Cellular edema has been known to increase epileptiform activity and seizure susceptibility in vitro and in vivo. However, the identity of the cell types undergoing volume increases and the types of excitability changes that occur in neurons remain unclear. Electrophysiological whole-cell patch clamp techniques were used to record currents and potentials from CA1 pyramidal neurons during the application of hypoosmolar ACSF (hACSF) in acutely isolated hippocampal slices from mice. Hypoosmolar ACSF evoked slow inward currents (SICs) in neurons, which initiated after ~1 minute of hACSF application. Neuronal excitability increased as osmolarity decreased in a dose-dependent manner. Even 5% reductions in osmolarity were sufficient to significantly increase neuronal excitability. In addition, hACSF induced neuronal firing of action potentials (APs), independent of AMPA receptor activation. Neuronal excitability was also increased during application of hACSF while blocking both APs and AMPA receptors. Increased sub-threshold EPSPs, neuronal APs, and bursting activity were also evoked in the presence of Mg2+, suggesting that hypoosmolar insults increase neuronal excitability under more physiological conditions. Hypoosmolar insults increased neuronal excitability in both juvenile (P15-P21) and adult 2- to 5-month-old) mice. Bursting activity in adult mice during osmotic insult was elevated compared to juvenile mice. During hypoosmolar insults the frequency of SICs recorded at physiological temperature were significantly elevated from SICs recorded at room temperature. SICs were potentiated by D-serine, and blocked by both DL-AP5 and the NR2B specific compound Ro25-6981. Together, these results indicate that osmotic insults produce cellular edema in both neurons and astrocytes, and increase neuronal excitability within minutes through a combination of synaptic and non-synaptic activation of glutamate receptors

Functional Effects of Cuprizone-Induced Demyelination in the Presence of the mTOR-Inhibitor Rapamycin

Persistent demyelination has been implicated in axon damage and functional deficits underlying neurodegenerative diseases such as multiple sclerosis. The cuprizone diet model of demyelination allows for the investigation of mechanisms underlying timed and reproducible demyelination and remyelination. However, spontaneous oligodendrocyte (OL) progenitor (OPC) proliferation, OPC differentiation, and axon remyelination during cuprizone diet may convolute the understanding of remyelinating events. The Akt (a serine/threonine kinase)/mTOR (the mammalian target of rapamycin) signaling pathway in OLs regulates intermediate steps during myelination. Thus, in an effort to inhibit spontaneous remyelination, the mTOR inhibitor rapamycin has been administered during cuprizone diet. Intrigued by the potential for rapamycin to optimize the cuprizone model by producing more complete demyelination, we sought to characterize the effects of rapamycin on axonal function and myelination. Functional remyelination was assessed by callosal compound action potential (CAP) recordings along with immunohistochemistry in mice treated with rapamycin during cuprizone diet. Rapamycin groups exhibited similar myelination, but significantly increased axonal damage and inflammation compared to non-rapamycin groups. There was minimal change in CAP amplitude between groups, however, a significant decrease in conduction velocity of the slower, non-myelinated CAP component was observed in the rapamycin group relative to the non-rapamycin group. During remyelination, rapamycin groups showed a significant decrease in OPC proliferation and mature OLs, suggesting a delay in OPC differentiation kinetics. In conclusion, we question the use of rapamycin to produce consistent demyelination as rapamycin increased inflammation and axonal damage, without affecting myelination

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca(2+) indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca(2+) events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease

Osmotic Edema Rapidly Increases Neuronal Excitability Through Activation of NMDA Receptor-Dependent Slow Inward Currents in Juvenile and Adult Hippocampus

Cellular edema (cell swelling) is a principal component of numerous brain disorders including ischemia, cortical spreading depression, hyponatremia, and epilepsy. Cellular edema increases seizure-like activity in vitro and in vivo, largely through nonsynaptic mechanisms attributable to reduction of the extracellular space. However, the types of excitability changes occurring in individual neurons during the acute phase of cell volume increase remain unclear. Using whole-cell patch clamp techniques, we report that one of the first effects of osmotic edema on excitability of CA1 pyramidal cells is the generation of slow inward currents (SICs), which initiate after approximately 1 min. Frequency of SICs increased as osmolarity decreased in a dose-dependent manner. Imaging of real-time volume changes in astrocytes revealed that neuronal SICs occurred while astrocytes were still in the process of swelling. SICs evoked by cell swelling were mainly nonsynaptic in origin and NMDA receptor-dependent. To better understand the relationship between SICs and changes in neuronal excitability, recordings were performed in increasingly physiological conditions. In the absence of any added pharmacological reagents or imposed voltage clamp, osmotic edema induced excitatory postsynaptic potentials and burst firing over the same timecourse as SICs. Like SICs, action potentials were blocked by NMDAR antagonists. Effects were more pronounced in adult (8–20 weeks old) compared with juvenile (P15–P21) mice. Together, our results indicate that cell swelling triggered by reduced osmolarity rapidly increases neuronal excitability through activation of NMDA receptors. Our findings have important implications for understanding nonsynaptic mechanisms of epilepsy in relation to cell swelling and reduction of the extracellular space

Bidirectional scaling of astrocytic metabotropic glutamate receptor signaling following long-term changes in neuronal firing rates.

Very little is known about the ability of astrocytic receptors to exhibit plasticity as a result of changes in neuronal activity. Here we provide evidence for bidirectional scaling of astrocytic group I metabotropic glutamate receptor signaling in acute mouse hippocampal slices following long-term changes in neuronal firing rates. Plasticity of astrocytic mGluRs was measured by recording spontaneous and evoked Ca²⁺ elevations in both astrocytic somata and processes. An exogenous astrocytic Gq G protein-coupled receptor was resistant to scaling, suggesting that the alterations in astrocyte Ca²⁺ signaling result from changes in activity of the surface mGluRs rather than a change in intracellular G protein signaling molecules. These findings suggest that astrocytes actively detect shifts in neuronal firing rates and adjust their receptor signaling accordingly. This type of long-term plasticity in astrocytes resembles neuronal homeostatic plasticity and might be important to ensure an optimal or expected level of input from neurons