Cortex Slices Research Articles

Recording of Local Field Potential in Mouse Hippocampal-Entorhinal Cortex Slices

Recording of Local Field Potential in Mouse Hippocampal-Entorhinal Cortex Slices

Recording of Local Field Potential in Mouse Hippocampal-Entorhinal Cortex Slices

Recording of Local Field Potential in Mouse Hippocampal-Entorhinal Cortex Slices

Effects of Anesthesia with Pentobarbital/Ketamine on Mitochondrial Permeability Transition Pore Opening and Ischemic Brain Damage.

The alteration of mitochondrial functions, especially the opening of the mitochondrial permeability transition pore (mPTP), has been proposed as a key mechanism in the development of lesions in cerebral ischemia, wherefore it is considered as an important target for drugs against ischemic injury. In this study, we aimed to investigate the effects of mitochondrial complex I inhibitors as possible regulators of mPTP using an in vitro brain ischemia model of the pentobarbital/ketamine (PBK)-anesthetized rats. We found that PBK anesthesia itself delayed Ca2+-induced mPTP opening and partially recovered the respiratory functions of mitochondria, isolated from rat brain cortex and cerebellum. In addition, PBK reduced cell death in rat brain slices of cerebral cortex and cerebellum. PBK inhibited the adenosine diphosphate (ADP)-stimulated respiration of isolated cortical and cerebellar mitochondria respiring with complex I-dependent substrates pyruvate and malate. Moreover, pentobarbital alone directly increased the resistance of isolated cortex mitochondria to Ca2+-induced activation of mPTP and inhibited complex I-dependent respiration and mitochondrial complex I activity. In contrast, ketamine had no direct effect on functions of isolated normal cortex and cerebellum mitochondria. Altogether, this suggests that modulation of mitochondrial complex I activity by pentobarbital during PBK anesthesia may increase the resistance of mitochondria to mPTP opening, which is considered the key event in brain cell necrosis during ischemia.

Dipeptide <i>L</i>-сarnosine (β-alanyl-<i>L</i>-histidine) — nervous tissue cryoprotector non-hybernate animals

In this work, the cryoprotective properties of dipeptide L-carnosine (β-alanyl-L-histidine) were studied on slices of the olfactory cortex of the brain of rats. Changes in the activity of N-methyl-D-aspartate receptors were analyzed as the most vulnerable to the effect of cryopreservation (CP), for this purpose, extracellular NMDA potentials were recorded. Slices were incubated with L-carnosine (20 mM) in the medium and frozen at a slow rate (0.1°C/min) down to –10°C and after CS (30 days) they were heated at the same rate (0.1°C/min) to +37°C. The effectiveness of cryoprotection of L-carnosine was determined by changes in the amplitudes of NMDA potentials after CP compared to before CP. The dipeptide restored the pH of the freezing medium 6.9 (without L-carnosine) to the optimum pH 7.3—7.4, promoted dehydration of free water from slices after CP, inhibited the development of glutamate excitotoxicity in slices. The data obtained prove that L-carnosine exhibits the properties of a non-toxic effective cryoprotector in the nervous tissue of warm-blooded non-hibernating animals.

INSPIRE: interpretable, flexible and spatially-aware integration of multiple spatial transcriptomics datasets from diverse sources.

Recent advances in spatial transcriptomics technologies have led to a growing number of diverse datasets, offering unprecedented opportunities to explore tissue organizations and functions within spatial contexts. However, it remains a significant challenge to effectively integrate and interpret these data, often originating from different samples, technologies, and developmental stages. In this paper, we present INSPIRE, a deep learning method for integrative analyses of multiple spatial transcriptomics datasets to address this challenge. With designs of graph neural networks and an adversarial learning mechanism, INSPIRE enables spatially informed and adaptable integration of data from varying sources. By incorporating non-negative matrix factorization, INSPIRE uncovers interpretable spatial factors with corresponding gene programs, revealing tissue architectures, cell type distributions and biological processes. We demonstrate the capabilities of INSPIRE by applying it to human cortex slices from different samples, mouse brain slices with complementary views, mouse hippocampus and embryo slices generated through different technologies, and spatiotemporal organogenesis atlases containing half a million spatial spots. INSPIRE shows superior performance in identifying detailed biological signals, effectively borrowing information across distinct profiling technologies, and elucidating dynamical changes during embryonic development. Furthermore, we utilize INSPIRE to build 3D models of tissues and whole organisms from multiple slices, demonstrating its power and versatility.

Mature oocyte found during ovarian tissue cryopreservation in an early adolescent female.

A 15-year-old female with Hodgkin's lymphoma underwent ovarian tissue cryopreservation for preserving fertility in Reproductive Department of Sir Run Run Shaw Hospital, Zhejiang University School of Medical after receiving one course of chemotherapy. During the ovarian tissue cryopreservation, one MⅡmature oocyte and three germinal vesicle oocytes were found. The three immature oocytes underwent in vitro maturation but failed. Ultimately, one mature oocyte and 12 ovarian cortex slices were cryopreserved using vitrification. This case indicates that for patients with established gonadal axis feedback, ovarian tissue cryopreservation may not be the only method for fertility preservation. It is advisable to consider ovarian stimulation and oocyte retrieval for oocyte cryopreservation. Alternatively, for individuals in the ovulation phase of their menstrual cycle, attempting oocyte retrieval before ovarian tissue cryopreservation to obtain mature oocytes from the natural cycle, followed by oocyte cryopreservation, may enhance the likelihood of successful fertility preservation.

Impaired synaptic function and hyperexcitability of the pyramidal neurons in the prefrontal cortex of autism-associated Shank3 mutant dogs

BackgroundSHANK3 gene is a highly replicated causative gene for autism spectrum disorder and has been well characterized in multiple Shank3 mutant rodent models. When compared to rodents, domestic dogs are excellent animal models in which to study social cognition as they closely interact with humans and exhibit similar social behaviors. Using CRISPR/Cas9 editing, we recently generated a dog model carrying Shank3 mutations, which displayed a spectrum of autism-like behaviors, such as social impairment and heightened anxiety. However, the neural mechanism underlying these abnormal behaviors remains to be identified.MethodsWe used Shank3 mutant dog models to examine possible relationships between Shank3 mutations and neuronal dysfunction. We studied electrophysiological properties and the synaptic transmission of pyramidal neurons from acute brain slices of the prefrontal cortex (PFC). We also examined dendrite elaboration and dendritic spine morphology in the PFC using biocytin staining and Golgi staining. We analyzed the postsynaptic density using electron microscopy.ResultsWe established a protocol for the electrophysiological recording of canine brain slices and revealed that excitatory synaptic transmission onto PFC layer 2/3 pyramidal neurons in Shank3 heterozygote dogs was impaired, and this was accompanied by reduced dendrite complexity and spine density when compared to wild-type dogs. Postsynaptic density structures were also impaired in Shank3 mutants; however, pyramidal neurons exhibited hyperexcitability.LimitationsCausal links between impaired PFC pyramidal neuron function and behavioral alterations remain unclear. Further experiments such as manipulating PFC neuronal activity or restoring synaptic transmission in Shank3 mutant dogs are required to assess PFC roles in altered social behaviors.ConclusionsOur study demonstrated the feasibility of using canine brain slices as a model system to study neuronal circuitry and disease. Shank3 haploinsufficiency causes morphological and functional abnormalities in PFC pyramidal neurons, supporting the notion that Shank3 mutant dogs are new and valid animal models for autism research.

Light-Driven Sodium Pump as a Potential Tool for the Control of Seizures in Epilepsy.

The marine flavobacterium Krokinobactereikastus light-driven sodium pump (KR2) generates an outward sodium ion current under 530nm light stimulation, representing a promising optogenetic tool for seizure control. However, the specifics of KR2 application to suppress epileptic activity have not yet been addressed. In the present study, we investigated the possibility of KR2 photostimulation to suppress epileptiform activity in mouse brain slices using the 4-aminopyrindine (4-AP) model. We injected the adeno-associated viral vector (AAV-PHP.eB-hSyn-KR2-YFP) containing the KR2 sodium pump gene enhanced with appropriate trafficking tags. KR2 expression was observed in the lateral entorhinal cortex and CA1 hippocampus. Using whole-cell patch clamp in mouse brain slices, we show that KR2, when stimulated with LED light, induces a substantial hyperpolarization of entorhinal neurons. However, continuous photostimulation of KR2 does not interrupt ictal discharges in mouse entorhinal cortex slices induced by a solution containing 4-AP. KR2-induced hyperpolarization strongly activates neuronal HCN channels. Consequently, turning off photostimulation resulted in HCN channel-mediated rebound depolarization accompanied by a transient increase in spontaneous network activity. Using low-frequency pulsed photostimulation, we induced the generation of short HCN channel-mediated discharges that occurred in response to the light stimulus being turned off; these discharges reliably interrupt ictal activity. Thus, low-frequency pulsed photostimulation of KR2 can be considered as a potential tool for controlling epileptic seizures.

Glutamate's Effects on the N-Methyl-D-Aspartate (NMDA) Receptor Ion Channel in Alzheimer's Disease Brain: Challenges for PET Radiotracer Development for Imaging the NMDA Ion Channel.

In an effort to further understand the challenges facing in vivo imaging probe development for the N-methyl-D-aspartate (NMDA) receptor ion channel, we have evaluated the effect of glutamate on the Alzheimer's disease (AD) brain. Human post-mortem AD brain slices of the frontal cortex and anterior cingulate were incubated with [3H]MK-801 and adjacent sections were tested for Aβ and Tau. The binding of [3H]MK-801 was measured in the absence and presence of glutamate and glycine. Increased [3H]MK-801 binding in AD brains was observed at baseline and in the presence of glutamate, indicating a significant increase (>100%) in glutamate-induced NMDA ion channel activity in AD brains compared to cognitively normal brains. The glycine effect was lower, suggesting a decrease of the co-agonist effect of glutamate and glycine in the AD brain. Our preliminary findings suggest that the targeting of the NMDA ion channel as well as the glutamate site may be appropriate in the diagnosis and treatment of AD. However, the low baseline levels of [3H]MK-801 binding in the frontal cortex and anterior cingulate in the absence of glutamate and glycine indicate significant hurdles for in vivo imaging probe development and validation.

Optogenetic stimulation suppresses ictal activity in a 4-aminopyridine model of epileptic activity <i>in vitro</i>

The WHO estimates that nearly 1% of the population suffers from epilepsy. Despite advances in the development of new antiepileptic drugs, seizures cannot be completely eliminated in nearly one-third of patients. One promising approach to the treatment of epilepsy may be gene therapy. Because epileptic activity is caused by an imbalance between excitation and inhibition, researchers have focused primarily on regulating neuronal excitability. Initially, the main approaches were based on the hyperexpression of inhibitory peptides such as galanin or NPY, or the suppression of neuronal excitability by the hyperexpression of potassium channels in neurons. However, these effects should be well calculated and strictly dosed, as it is difficult to correct the expression later. If the expression is too low, the anticonvulsant effect will not be achieved, and if the expression is too high, the neuronal networks will be impaired due to strong inhibition. For this reason, methodological approaches to treatment in which the effect on the neuronal excitability in the epileptic focus can be controlled are of great interest. Optogenetic methods offer such an advantage. Optogenetics uses light to alter the excitability of specific neuronal populations and can also be used in a biofeedback paradigm in which the light source is activated only at the risk of generating seizure activity. A number of optogenetic tools have now been developed, including light-activated cationic (e.g., ChR2) and anion channels (ACR), metabotropic receptors, pumps (NpHR, Arch), and enzymes. However, the optogenetic approach has a number of technical difficulties in delivering the light source and the risk of developing an immune response to the expression of rhodopsins. This report reviews the experience of practical application of optogenetic tools in the use of 4-aminopyridine in vitro model of epileptiform activity in experiencing slices of the entorhinal cortex. We tested the efficacy of suppressing ictal activity using several variants of optogenetic stimulation: (1) activation of excitatory and inhibitory neurons (in Thy1-ChR2-YFP mice), (2) activation of inhibitory interneurons only (in PV-Cre mice after virus injection with the channelodopsin-2 gene), hyperpolarization of excitatory neurons after expression of (3) archaeodopsin or (4) a light-dependent sodium pump. We found that ictal activity was successfully suppressed when low-frequency optogenetic stimulation induced regular interictal activity. Usually, interictal activity was induced by rhythmic synchronous activation of the principal neurons of the entorhinal cortex. In other cases, the ictal activity was preserved, although its characteristics may have changed. We determined the parameters of optogenetic stimulation that were most effective in suppressing ictal activity. The availability of a wide range of gene therapy approaches for epilepsy that have demonstrated efficacy in preclinical studies suggests that clinical trials of some of these approaches will begin in the next few years.

Cholinergic Activation of Corticofugal Circuits in the Adult Mouse Prefrontal Cortex.

Acetylcholine (ACh) promotes neocortical output to the thalamus and brainstem by preferentially enhancing the postsynaptic excitability of layer 5 pyramidal tract (PT) neurons relative to neighboring intratelencephalic (IT) neurons. Less is known about how ACh regulates the excitatory synaptic drive of IT and PT neurons. To address this question, spontaneous excitatory postsynaptic potentials (sEPSPs) were recorded in dual recordings of IT and PT neurons in slices of prelimbic cortex from adult female and male mice. ACh (20 µM) enhanced sEPSP amplitudes, frequencies, rise-times, and half-widths preferentially in PT neurons. These effects were blocked by the muscarinic receptor antagonist atropine (1 µM). When challenged with pirenzepine (1 µM), an antagonist selective for M1-type muscarinic receptors, ACh instead reduced sEPSP frequencies, suggesting that ACh may generally suppress synaptic transmission in the cortex via non-M1 receptors. Cholinergic enhancement of sEPSPs in PT neurons was not sensitive to antagonism of GABA receptors with gabazine (10 µM) and CGP52432 (2.5 µM) but was blocked by tetrodotoxin (1 µM), suggesting that ACh enhances action-potential-dependent excitatory synaptic transmission in PT neurons. ACh also preferentially promoted the occurrence of synchronous sEPSPs in dual recordings of PT neurons relative to IT-PT and IT-IT parings. Finally, selective chemogenetic silencing of hM4Di-expressing PT, but not commissural IT, neurons blocked cholinergic enhancement of sEPSP amplitudes and frequencies in PT neurons. These data suggest that, in addition to selectively enhancing the postsynaptic excitability of PT neurons, M1 receptor activation promotes corticofugal output by amplifying recurrent excitation within networks of PT neurons.

Integrating spatial transcriptomics data across different conditions, technologies and developmental stages.

With the rapid generation of spatial transcriptomics (ST) data, integrative analysis of multiple ST datasets from different conditions, technologies and developmental stages is becoming increasingly important. Here we present a graph attention neural network called STAligner for integrating and aligning ST datasets, enabling spatially aware data integration, simultaneous spatial domain identification and downstream comparative analysis. We apply STAligner to ST datasets of the human cortex slices from different samples, the mouse olfactory bulb slices generated by two profiling technologies, the mouse hippocampus tissue slices under normal and Alzheimer's disease conditions, and the spatiotemporal atlases of mouse organogenesis. STAligner efficiently captures the shared tissue structures across different slices, the disease-related substructures and the dynamical changes during mouse embryonic development. In addition, the shared spatial domain and nearest-neighbor pairs identified by STAligner can be further considered as corresponding pairs to guide the three-dimensional reconstruction of consecutive slices, achieving more accurate local structure-guided registration than the existing method.

Role of DAG Signaling Pathway in Bipolar Disorder

Lithium is an effective mood stabilizer in 70-80 percent of bipolar disorder patients. It has been observed that the membrane potential is hyperpolarized in bipolar patients and lithium treatment depolarizes it. Hokin and his colleagues found that the lithium increases accumulation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) in brain cortex slices in species ranging from mouse to monkey. DAG and IP3 activate the DAG signaling pathway depolarizing the membrane potential. The DAG signaling pathway regulates the calcium activated potassium (CAK) channels. The CAK channels in turn control the membrane potentials and excitabilities of neurons. The clinical observations that the membrane potential is hyperpolarized (lower) in bipolar disorder patients and lithium depolarizes (increases) are explained using the action of DAG signaling pathway. Lithium increases the concentration of DAG thereby increasing the activity of DAG signaling pathway which controls the potassium flow through the CAK channel. If this result is established through further independent corroboration it would greatly enhance our understanding of the mechanisms of bipolar disorder. Knowledge of this signaling pathway elucidates how lithium treats bipolar disorder. Additionally, the mechanism of induced switching associated with certain antidepressants given to patients who are bipolar can also be explained by this pathway. Further analysis of this pathway would lead to the discovery of new drugs for this disease.

Probucol neuroprotection against manganese-induced damage in adult Wistar rat brain slices.

Manganese (Mn) is an abundant element used for commercial purposes and is essential for the proper function of biological systems. Chronic exposure to high Mn concentrations causes Manganism, a Parkinson's-like neurological disorder. The pathophysiological mechanism of Manganism remains unknown; however, it involves mitochondrial dysfunction and oxidative stress. This study assessed the neuroprotective effect of probucol, a hypolipidemic agent with anti-inflammatory and antioxidant properties, on cell viability and oxidative stress in slices of the cerebral cortex and striatum from adult male Wistar rats. Brain structure slices were kept separately and incubated with manganese chloride (MnCl2) and probucol to evaluate the cell viability and oxidative parameters. Probucol prevented Mn toxicity in the cerebral cortex and striatum, as evidenced by the preservation of cell viability observed with probucol (10 and 30μM) pre-treatment, as well as the prevention of mitochondrial complex I inhibition in the striatum (30μM). These findings support the protective antioxidant action of probucol, attributed to its ability to prevent cell death and mitigate Mn-induced mitochondrial dysfunction.

Tissue oxygen partial pressure as a viability metric for ex vivo brain tissue slices

BackgroundDespite the prevalent use of the ex vivo brain slice preparation in neurophysiology research, a reliable method for judging tissue viability ― and thus suitability of a slice for inclusion in an experiment ― is lacking. The utility of indirect electrophysiological measures of tissue health is model-specific and needs to be used cautiously. In this study, we verify a more direct test of slice viability, based on tissue oxygen consumption rate. New methodWe hypothesised that the minimum intra-slice partial pressure of oxygen (pO2min) would correlate with tissue oxygen consumption rate, providing an accessible method for reliably assessing tissue viability status. Using mouse brain cortex slices, we measured tissue oxygen consumption rate using a Fick’s law diffusion–consumption model applied to full intra-tissue pO2 profiles and compared this to pO2min and 2,3,5-triphenol tetrazolium chloride (TTC) viability staining. ResultsTissue pO2min correlated strongly with oxygen consumption rate in both neurophysiological active and quiescent tissue (in “no-magnesium” and “normal” artificial cerebrospinal fluid, respectively) (R2 =49.7% and 42.1%, respectively). Both correlated with TTC viability stain. Oxygen consumption rate was positively related to the frequency of seizure-like event activity in no-magnesium artificial cerebrospinal fluid (R2 = 44.8%). Comparison with existing methodsWhile measurement of tissue oxygen levels and oxygen consumption is not new, intra-tissue pO2min is a novel approach to assess brain slice viability. ConclusionThe results confirm that tissue oxygen minimum pO2min is a robust metric for estimating tissue viability status — the lower the pO2min, the healthier the tissue.

The volatile anesthetic isoflurane differentially inhibits voltage-gated sodium channel currents between pyramidal and parvalbumin neurons in the prefrontal cortex.

How volatile anesthetics work remains poorly understood. Modulations of synaptic neurotransmission are the direct cellular mechanisms of volatile anesthetics in the central nervous system. Volatile anesthetics such as isoflurane may reduce neuronal interaction by differentially inhibiting neurotransmission between GABAergic and glutamatergic synapses. Presynaptic voltage-dependent sodium channels (Nav), which are strictly coupled with synaptic vesicle exocytosis, are inhibited by volatile anesthetics and may contribute to the selectivity of isoflurane between GABAergic and glutamatergic synapses. However, it is still unknown how isoflurane at clinical concentrations differentially modulates Nav currents between excitatory and inhibitory neurons at the tissue level. In this study, an electrophysiological recording was applied in cortex slices to investigate the effects of isoflurane on Nav between parvalbumin (PV+) and pyramidal neurons in PV-cre-tdTomato and/or vglut2-cre-tdTomato mice. Isoflurane at clinically relevant concentrations produced a hyperpolarizing shift in the voltage-dependent inactivation and slowed the recovery time from the fast inactivation in both cellular subtypes. Since the voltage of half-maximal inactivation was significantly depolarized in PV+ neurons compared to that of pyramidal neurons, isoflurane inhibited the peak Nav currents in pyramidal neurons more potently than those of PV+ neurons (35.95 ± 13.32% vs. 19.24 ± 16.04%, P = 0.036 by the Mann-Whitney test). Isoflurane differentially inhibits Nav currents between pyramidal and PV+ neurons in the prefrontal cortex, which may contribute to the preferential suppression of glutamate release over GABA release, resulting in the net depression of excitatory-inhibitory circuits in the prefrontal cortex.

Agar freezing solution for long-term cryopreservation of brain slices from non-hibernating animals

Our previous studies have shown that function of ionotropic glutamate receptors such as AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) and NMDA (N-methyl-D-aspartate) was impaired after long-term cryopreservation of brain slices at -10°C within 30-50 days. To elucidate the reasons for cryodamage to AMPA- and NMDA-dependent mechanisms, artificial cerebrospinal fluid (aCSF) solutions that contain agar at different concentrations (33, 44, and 50%) were used for cryopreservation of the rat olfactory cortex slices. After cryopreservation, the slices were warmed to 37°C and the amplitudes changes of AMPA and NMDA potentials, which reflected the activities of the AMPA and NMDA mechanisms, were evaluated; the results were compared with those obtained before cryopreservation. It was found that AMPA and NMDA potentials changed differently depending on the concentration of agar in artificial cerebrospinal fluid. In solutions with 33% agar, the amplitude of AMPA potentials increased by 60%, whereas, in contrast, the amplitude of NMDA potentials was equal to the values before cryopreservation. At agar concentration of 44% in the solution, the AMPA and NMDA amplitudes were increased by 70% and 80%, respectively. A complete recovery of the activities of AMPA and NMDA mechanisms was obtained after cryopreservation in a freezing solution with an agar concentration of 50%. Under these conditions, the amplitudes of the AMPA and NMDA potentials corresponded to those seen before cryopreservation. Thus, the results obtained indicate that agar added to the artificial cerebrospinal fluid solution is a cryoprotectant that protects AMPA- and NMDA-dependent mechanisms from cryoinjury. The freezing solution (artificial cerebrospinal fluid and agar) developed by us for cryopreservation of brain explants of non-hibernating animals will be used to create a cryobank of nervous tissue.

Rescue of synaptic and cognitive functions in polysialic acid-deficient mice and dementia models by short polysialic acid fragments

Dysregulated cortical expression of the neural cell adhesion molecule (NCAM) and deficits of its associated polysialic acid (polySia) have been found in Alzheimer's disease and schizophrenia. However, the functional role of polySia in cortical synaptic plasticity remains poorly understood. Here, we show that acute enzymatic removal of polySia in medial prefrontal cortex (mPFC) slices leads to increased transmission mediated by the GluN1/GluN2B subtype of N-methyl-d-aspartate receptors (NMDARs), increased NMDAR-mediated extrasynaptic tonic currents, and impaired long-term potentiation (LTP). The latter could be fully rescued by pharmacological suppression of GluN1/GluN2B receptors, or by application of short soluble polySia fragments that inhibited opening of GluN1/GluN2B channels. These treatments and augmentation of synaptic NMDARs with the glycine transporter type 1 (GlyT1) inhibitor sarcosine also restored LTP in mice deficient in polysialyltransferase ST8SIA4. Furthermore, the impaired performance of polySia-deficient mice and two models of Alzheimer's disease in the mPFC-dependent cognitive tasks could be rescued by intranasal administration of polySia fragments. Our data demonstrate the essential role of polySia-NCAM in the balancing of signaling through synaptic/extrasynaptic NMDARs in mPFC and highlight the therapeutic potential of short polySia fragments to restrain GluN1/GluN2B-mediated signaling.

Anoxic spreading depolarization in the neonatal rat cortex in vitro.

Anoxic spreading depolarization (aSD) is a hallmark of ischemic injury in the cerebral cortex. In adults, aSD is associated with rapid and nearly complete neuronal depolarization and loss of neuronal functions. While ischemia also evokes aSD in the immature cortex, developmental aspects of neuronal behavior during aSD remain largely unknown. Here, using oxygen-glucose deprivation (OGD) ischemia model in slices of the postnatal rat somatosensory cortex, we found that immature neurons displayed much more complex behaviors: they initially moderately depolarized during aSD, then transiently repolarised (for up to tens of minutes), and only then passed to terminal depolarization. The ability to fire action potentials was maintained in neurons mildly depolarized during aSD without reaching the level of depolarization block, and these functions were regained in the majority of immature neurons during post-aSD transient repolarization. The amplitude of depolarization and the probability of depolarization block during aSD increased, whereas transient post-SD repolarization levels and duration, and associated recovery in neuronal firing decreased with age. By the end of the first postnatal month, aSD acquired an adult-like phenotype, where depolarization during aSD merged with terminal depolarization and the phase of transient recovery was lost. Thus, changes in neuronal function during aSD undergo remarkable developmental changes that may contribute to lower susceptibility of the immature neurons to ischemia.

Cryoprotective Characteristics of L-Carnosine Dipeptide (β-Alanyl-L-Histidine)

The dipeptide (β-alanyl-L-histidine) is found in significant amounts in the muscles and brain of mammals, especially in the olfactory structures. L-Carnosine exhibits many protective effects when exposed to various cytotoxic factors on cells. We used slices of the rat olfactory cortex to study the cryoprotective characteristics of L-carnosine during cryopreservation (CP). Changes in the activity of N-methyl-D-aspartate receptors (NMDAR) were analyzed during registration of NMDA potentials induced by electrical stimulation of the lateral olfactory tract. Brain slices were preincubated with L-carnosine (20 mM) in solution, frozen (−10°C), and after a long CP (30 days) they were warmed up to 37°C and changes in the amplitudes of NMDA potentials were determined. It was found that the dipeptide optimized the pH of the freezing solution after CP and retained the activity of NMDAR, determined by the amplitude of NMDA potentials. L-Carnosine after CP contributed to the dehydration of excess free water from the slices. The dipeptide inhibited the development of glutamate excitotoxicity in brain slices during CP and maintained normal NMDAR functioning. The data obtained prove that L-carnosine exhibits the properties of an endogenous cryoprotector in the nervous tissue.