Adult Slices Research Articles

An in vitro spinal cord slice preparation for recording from lumbar motoneurons of the adult mouse

The development of central nervous system slice preparations for electrophysiological studies has led to an explosion of knowledge of neuronal properties in health and disease. Studies of spinal motoneurons in these preparations, however, have been largely limited to the early postnatal period, as adult motoneurons are vulnerable to the insults sustained by the preparation. We therefore sought to develop an adult spinal cord slice preparation that permits recording from lumbar motoneurons. To accomplish this, we empirically optimized the composition of solutions used during preparation in order to limit energy failure, reduce harmful ionic fluxes, mitigate oxidative stress, and prevent excitotoxic cell death. In addition to other additives, this involved the use of ethyl pyruvate, which serves as an effective nutrient and antioxidant. We also optimized and incorporated a host of previously published modifications used for other in vitro preparations, such as the use of polyethylene glycol. We provide an in-depth description of the preparation protocol and discuss the rationale underlying each modification. By using this protocol, we obtained stable whole cell patch-clamp recordings from identified fluorescent protein-labeled motoneurons in adult slices; here, we describe the firing properties of these adult motoneurons. We propose that this preparation will allow further studies of how motoneurons integrate activity to produce adult motor behaviors and how pathological processes such as amyotrophic lateral sclerosis affect these neurons.

Periadolescent changes of D2–AMPA interactions in the rat nucleus accumbens

Many aspects of dopamine (DA) systems mature during adolescence. In the nucleus accumbens, the modulation of prefrontal cortical synaptic responses by DA becomes refined during adolescence with the recruitment of a gamma-amino butyric acid (GABA) component. As this GABA component is depolarizing, it remains to be determined whether this change affects action potential firing in nucleus accumbens neurons. Here we tested whether a D(2) agonist affects AMPA-evoked cell firing in slices containing the nucleus accumbens from juvenile (postnatal day, PD 28-34) and adult (PD > 60) rats. 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) (0.1-0.4 μM) depolarized nucleus accumbens neurons and increased their firing in a dose-dependent manner. The D(2) agonist quinpirole (2 μM) had different effects in juvenile vs. adult slices. In the juvenile accumbens, quinpirole enhanced AMPA (0.2 μM) effects on evoked firing in a subset of neurons while it had no effect on the rest. In the adult accumbens, the D(2) agonist instead attenuated the effect of AMPA on evoked firing, an interaction that was blocked by the GABA-A antagonist picrotoxin (50 μM). Thus, D(2) receptors modulate AMPA responses in the nucleus accumbens differently in juvenile than adult rats, and the adult effect requires local GABA transmission. The incorporation of a GABA component in the modulation of information processing in the nucleus accumbens by DA during adolescence may allow for a better contrast in cortically activated ensembles.

Long-term patch recordings from adult spinal neurons herald new era of opportunity

Recently, Andreas Husch, Nathan Cramer, and Ronald M. Harris-Warrick achieved a remarkable breakthrough in patch-clamp recordings of ventral horn neurons in the adult spinal cord slice preparation. This landmark study that breaks the "age barrier" is titled "Long-duration perforated patch recordings from spinal interneurons of adult mice" (Husch et al., in press). In it, the authors demonstrate the unprecedented ability to undertake day-long (up to 12 h), and utterly stable perforated patch recordings. A description of the methodology is detailed in their paper. Here, I give a brief overview before providing context to this extraordinary achievement.

Mitral Cells of the Olfactory Bulb Perform Metabolic Sensing and Are Disrupted by Obesity at the Level of the Kv1.3 Ion Channel

Sixty-five percent of Americans are over-weight. While the neuroendocrine controls of energy homeostasis are well known, how sensory systems respond to and are impacted by obesity is scantily understood. The main accepted function of the olfactory system is to provide an internal depiction of our external chemical environment, starting from the detection of chemosensory cues. We hypothesized that the system additionally functions to encode internal chemistry via the detection of chemicals that are important indicators of metabolic state. We here uncovered that the olfactory bulb (OB) subserves as an internal sensor of metabolism via insulin-induced modulation of the potassium channel Kv1.3. Using an adult slice preparation of the olfactory bulb, we found that evoked neural activity in Kv1.3-expressing mitral cells is enhanced following acute insulin application. Insulin mediated changes in mitral cell excitability are predominantly due to the modulation of Kv1.3 channels as evidenced by the lack of effect in slices from Kv1.3-null mice. Moreover, a selective Kv1.3 peptide blocker (ShK186) inhibits more than 80% of the outward current in parallel voltage-clamp studies, whereby insulin significantly decreases the peak current magnitude without altering the kinetics of inactivation or deactivation. Mice that were chronically administered insulin using intranasal delivery approaches exhibited either an elevation in basal firing frequency or fired a single cluster of action potentials. Following chronic administration of the hormone, mitral cells were inhibited by application of acute insulin rather than excited. Mice made obese through a diet of ∼32% fat exhibited prominent changes in mitral cell action potential shape and clustering behavior, whereby the subsequent response to acute insulin stimulation was either attenuated or completely absent. Our results implicate an inappropriate neural function of olfactory sensors following exposure to chronic levels of the hormone insulin (diabetes) or increased body weight (obesity).

Evaluation of cell damage in organotypic hippocampal slice culture from adult mouse: A potential model system to study neuroprotection

The use of organotypic hippocampal slice culture (OHSC) has become a powerful tool for studying cell damage in different neuropathological states, since it reproduces the basic morphological and functional properties of hippocampal neuronal network. However, the conventional OHSCs are established from postnatal animals rather than adult. Here we reevaluated the features of cell death in adult OHSC in detail and found potential utility for the study of neuroprotection. Organotypic culture of hippocampal slices from adult mice under conventional conditions led to a time-dependent and reproducible cell death. Around 6days in vitro (DIV), slices lost 50% of the cells, based on LDH release assessment. The cell death was greater than 90% after DIV 15. The cell loss was linearly correlated (r=0.944, P<0.01) with the time in culture. The electrophysiological responses to the stimulus in the cultured adult slices were accordingly reduced. The cell degeneration during adult OHSC might be utilized as a tool for studying neuroprotective effects in drug development. To illustrate this potential use, adult OHSCs were challenged with brain-derived neurotrophic factor (BDNF). We found that the continuous supplementation of 300ng/ml BDNF promoted cell survival of adult OHSC. Using immunohistochemistry and Western blot analyses of neuronal markers, we also demonstrated the pro-survival effects of BDNF on neurons in the adult OHSC system. It is suggested that OHSCs from adult mice might provide an alternative model system for neuronal degeneration, suitable for studying physiological factors and pharmacological compounds contributing to neuronal survival.

The binding site for acute corticosterone effects on &amp;lt;i&amp;gt;N&amp;lt;/i&amp;gt;-methyl-D-aspartate receptor-mediated Ca&amp;lt;sup&amp;gt;2+&amp;lt;/sup&amp;gt; signals in mouse hippocampal slices

We have examined acute effects of corticosterone (CORT) on N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ signals in adult mouse hippocampal slices. We found so far that the 30 min preincubation of CORT induced a signifcant decrease of the peak amplitude of NMDA-induced Ca2+ elevation in the CA1 region. The membrane non-permeable bovine serum albumin-conjugated CORT also induced a similar effect in the CA1 region. Therefore the acute CORT effects should be induced via putative surface CORT receptors. A possible candidate is a classical intracellular glucocorticoid receptor (GR). To confirm this speculation, we here examined the effects of dexamethasone (DEX: an agonist of GR) and CORT with RU38486 (an antagonist of GR) on NMDA-induced Ca2+ signals. As a result, DEX induced a similar effect to the suppressive CORT effect in the CA1 region, and RU38486 inhibited the suppressive CORT effect. These results indicate that the surface CORT receptor should be GR bound to plasma mem- brane.

177. ANTIBODIES AGAINST BMPR-IB UNCOVERS A PARACRINE FUNCTION FOR THE RECEPTOR IN MALE MOUSE LEYDIG CELL TESTOSTERONE PRODUCTION IN VITRO

The male reproductive system is regulated by pituitary gonadotrophins and local testicular factors including the transforming growth factor-β superfamily members which up-regulate and modulate testosterone production respectively. Type I and type II bone morphogenetic protein (BMP) receptors have been identified in both steroidogenic and non-steroidogenic cells in the testis and have been reported to impact steroid production via their effect on steroidogenic enzymes. In this study we investigated if BMPR-IB had an autocrine or paracine role in testosterone production using BMPR-IB antibodies in testis tissue and Leydig cell culture in vitro. Immature (21d) and mature (60d) mice were sacrificed by CO2 asphyxiation and the testis removed, decapsulated and cultured in basal and equine chorionic gonadotrophin (eCG)-conditioned media for three hours at 32 °C in 5 % CO2, in the presence and absence of anti-BMPR-IB. Additionally, Leydig cells were Percoll purified from adult mouse testicular interstitial cells and cultured for three hours with and without anti-BMPR-IB under human chorionic gonadotrophin (hCG) stimulated or unstimulated conditions. After three hours of incubation the culture media was aspirated into labeled vials and assayed for testosterone using a radioimmunoassay. In adult testicular slice culture, treatment with anti-BMPR-IB resulted in a significant decrease in basal and eCG-stimulated testosterone production by 37 % and 41 % respectively, while having no significant effect on basal or eCG-stimulated testosterone production by the immature testis. In purified Leydig cell culture from adult male mice BMPR-IB immunization had no effect on testosterone production in basal or hCG-stimulated conditions. In conclusion, anti-BMPR-IB significantly reduced testosterone production in adult testicular slice culture but not in cell culture, demonstrating that BMP paracrine signalling from the seminiferous tubules is likely to be important in modulating testosterone production by Leydig cells. Additionally, the paracrine signalling appears to be developmentally regulated only occurring in the adult testis.

Evidence against AMPA Receptor-Lacking Glutamatergic Synapses in the Superficial Dorsal Horn of the Rat Spinal Cord

Pure NMDA receptor (NMDAr)-mediated EPSCs, thought to correspond to "silent" glutamatergic synapses that lack AMPA receptors (AMPArs), have been observed in superficial spinal dorsal horn of neonatal but not adult rats. Recent anatomical studies suggest that AMPArs are present at virtually all glutamatergic synapses in this region in adults. We used antigen retrieval to examine colocalization of AMPArs and PSD-95 (a marker for glutamatergic synapses) in laminae I-II of neonatal and adult rats. We found a high degree of colocalization in all cases, which suggests that AMPArs are present in the great majority of glutamatergic synapses even in neonatal animals. We therefore reexamined evidence for silent synapses by performing blind whole-cell recordings from superficial dorsal horn neurons in slices from neonatal or adult rats, with focal stimulation to activate glutamatergic synapses. On some occasions in both neonatal (10 of 109, 9%) and adult (9 of 77, 12%) slices, NMDAr-mediated EPSCs were observed when the holding potential was raised to +50 mV at a stimulus strength that had failed to evoke AMPAr-mediated EPSCs. However, in all cases tested, AMPAr-mediated EPSCs were then observed when the cell was returned to -70 mV; this and other properties of the EPSCs suggest that they do not represent genuine silent synapses. When compared with previous findings, our results indicate that the appearance of silent synapses depends on experimental protocol. This suggests that pure NMDAr-mediated EPSCs seen in previous studies do not correspond to AMPAr-lacking synapses but result from another mechanism, for example, loss of labile AMPArs from recently formed synapses.

A mouse juvenile or adult slice with preserved functional nigro-striatal dopaminergic neurons

The effect of endogenous dopamine on the activity of target neurons recorded with patch clamp or Ca2+ imaging techniques in slices has been studied to date with intra-striatal stimuli. Yet, this approach is severely handicapped by the nonphysiological and nonspecific stimulation of local neurons and fibers within the striatum. We now report a new juvenile and adult mouse slice preparation in which a component of the nigro-striatal dopaminergic pathway is preserved in its entirety, from cell bodies to axon terminals. This tilted parasagittal slice (380–400 μm) just medial to the subthalamic nucleus contains functional nigro-striatal neurons as assessed by morphological examination of tyrosine hydroxylase positive cell bodies and axons, combined with electrochemical assays of dopamine release in the striatum in response to stimulation of the substantia nigra pars compacta. The nigro-striatal slice constitutes a suitable in vitro preparation to determine the impact of endogenously released dopamine on target neurons of the striatum.

LACK OF CELLULAR PRION PROTEIN UNMASKS NMDA NR2D SUBUNIT RECEPTOR FUNCTION WITH CONSEQUENCES TOWARD SYNAPTIC TRANSMISSION AND EXCITOTOXICITY

Background: The physiological functions of endogenous cellular prion protein (PrPC)is incompletely understood. Previously, it has been shown that PrP-null mice exhibit reduced long-term (synaptic) potentiation and greater susceptibility to seizure mortality in several in vivo models of epilepsy. In addition, PrP-null neurons in culture exhibit greater excito toxic cell death in response to kainic acid exposure, and in several models of oxidative stress. Although PrP seems toplay a protective role against various forms of cellular insults, the precise mechanism of such action is unknown. Methods: We investigated the synaptic properties of WT and PrP-null mice using cultured neurons and also brain slices from adult mice. Synaptic activity was assessed using whole-cell voltage clamp. We recorded spontaneous and evoked synaptic potentials. Extracellular field recordings of brain slices were also performed. Pharmacological agents were used to isolate all components of glutamatergic and GABA(A) mediated synaptic transmission. In addition, weassessed the effect of NMDA excitotoxicity in WT and PrP-null neurons using in vitro and in vivo experiments. We also used immunostaining, coimmunoprecipitation, and protein expression studies to quantify the relation between NMDA subtype expression and localization relative to native PrP. Results: Recordings in the CA1 layer of adult hippocampal slices showed thatPrP-null mice exhibit a reduced threshold to evoked responses, exhibited basal hyperexcitability, and in a model of zero-Mg2+ seizures also showed lower seizure threshold. No differences were observed in paired-pulse facilitation relative to WT animals. Recordings from mature hippocampal cultures showed slightly altered AMPA and GABAA miniature synaptic currents. NMDA mEPSCs were observed to be increased in amplitude and significantly prolonged in decaytime. NMDA-evoked currents also exhibited markedly prolonged deactivation kinetics. This effect on evoked NMDA currents was reproduced in WT neurons byindependent PrP-RNAi, NR2D-RNAi transfection, and eliminated by PrPCtransfection into PrP-null neurons. In addition, PrP coimmunoprecipitated with NR2D and not NR2B NMDA receptor subunits. In vitro and in vivo experiments utilizing transient exposure to NMDA showed greater cell death in PrP-nullneurons, which was significantly reduced by application of an NMDA receptor antagonist. Conclusions: These data suggest that enhanced NMDA activity is present in PrP-null neurons. Consistent with this finding, in vitro and in vivo excitotoxicity assays demonstrated increased neuronal cell death in PrP-null cultures and animals upon transient exposure to NMDA. This was confirmed at the level of synaptic currents showing prolonged receptor deactivation kinetics that were most consistent with functional activation of NR2D NMDA receptor subunits. Enhanced NMDA receptor function was paralleled by increased excitotoxicity in PrP-null mice. Our findings demonstrate a novel functional role for PrP as a modulator of synaptic NMDA currents and attributes a neuroprotective function to PrP.

Differential Induction of Long-Term Potentiation in the Horizontal versus Columnar Superficial Connections to Layer II Cells of the Entorhinal Cortex

The entorhinal cortex (EC) is a nodal and independent mnemonic element of the medial temporal lobe memory circuit as it forms a bidirectional interface between the neocortex and hippocampus. Within the EC, intra- and inter-lamellar associational connections occur via horizontal and columnar projections, respectively. We undertook a comparative study of these two inputs as they converge upon EC layer II cells using whole-cell patch techniques in an adult rat EC horizontal slice preparation in which the deepest layers (V-VI) had been dissected out. Electrical stimulation of layers I and III during GABA blockade allowed us to study excitatory synaptic properties and plasticity in the horizontal and columnar fibre systems, respectively. Both pathways exhibited AMPA- and NMDA-receptor mediated transmission and both exhibited long-term potentiation (LTP) after high-frequency (tetanic) stimulation. LTP in the horizontal, but not in the columnar pathway, was blocked by NMDA receptor antagonism. Intriguingly, LTP in both appeared to be mediated by post synaptic increases in Ca2+ that may be coupled to differing second messenger pathways. Thus, the superficial excitatory horizontal and columnar associative pathways to layer II have divergent mechanisms for LTP which may endow the EC with even more complex and dynamic processing characteristics than previously thought.

Group III mGluR regulation of synaptic transmission at the SC-CA1 synapse is developmentally regulated

Group III metabotropic glutamate receptors (mGluRs) reduce synaptic transmission at the Schaffer collateral-CA1 (SC-CA1) synapse in rats by a presynaptic mechanism. Previous studies show that low concentrations of the group III-selective agonist, L-AP4, reduce synaptic transmission in slices from neonatal but not adult rats, whereas high micromolar concentrations reduce transmission in both age groups. L-AP4 activates mGluRs 4 and 8 at much lower concentrations than those required to activate mGluR7, suggesting that the group III mGluR subtype modulating transmission is a high affinity receptor in neonates and a low affinity receptor in adults. The previous lack of subtype selective ligands has made it difficult to test this hypothesis. We have measured fEPSPs in the presence of novel subtype selective agents to address this question. We show that the effects of L-AP4 can be blocked by LY341495 in both neonates and adults, verifying that these effects are mediated by mGluRs. In addition, the selective mGluR8 agonist, DCPG, has a significant effect in slices from neonatal rats but does not reduce synaptic transmission in adult slices. The mGluR4 selective allosteric potentiator, PHCCC, is unable to potentiate the L-AP4-induced effects at either age. Taken together, our data suggest that group III mGluRs regulate transmission at the SC-CA1 synapse throughout development but there is a developmental regulation of the subtypes involved so that both mGluR7 and mGluR8 serve this role in neonates whereas mGluR7 is involved in regulating transmission at this synapse throughout postnatal development.

Cholinergic modulation of intrinsic fibre-evoked excitatory transmission contains a nicotinic component in immature but not adult rat piriform cortex, in vitro

The piriform cortex (PC) is highly prone to epileptogenesis, particularly in immature animals, where decreased muscarinic modulation of PC intrinsic fibre excitatory neurotransmission is implicated as a likely cause. However, whether higher levels of acetylcholine (ACh) release occur in immature vs. adult PC remains unclear. We investigated this using in vitro extracellular electrophysiological recording techniques. Intrinsic fibre-evoked extracellular field potentials (EFPs) were recorded from layers II to III in PC brain slices prepared from immature (P14-18) and adult (P>40) rats. Adult and immature PC EFPs were suppressed by eserine (1 microM) or neostigmine (1 microM) application, with a greater suppression in immature (approximately 40%) than adult (approximately 30%) slices. Subsequent application of atropine (1 microM) reversed EFP suppression, producing supranormal (approximately 12%) recovery in adult slices, suggesting that suppression was solely muscarinic ACh receptor-mediated and that some 'basal' cholinergic 'tone' was present. Conversely, atropine only partially reversed anticholinesterase effects in immature slices, suggesting the presence of additional non-muscarinic modulation. Accordingly, nicotine (50 microM) caused immature field suppression (approximately 30%) that was further enhanced by neostigmine, whereas it had no effect on adult EFPs. Unlike atropine, nicotinic antagonists, mecamylamine and methyllycaconitine, induced immature supranormal field recovery (approximately 20%) following anticholinesterase-induced suppression (with no effect on adult slices), confirming that basal cholinergic 'tone' was also present. We suggest that nicotinic inhibitory cholinergic modulation occurs in the immature rat PC intrinsic excitatory fibre system, possibly to complement the existing, weak muscarinic modulation, and could be another important developmentally regulated system governing immature PC susceptibility towards epileptogenesis.

Modulation of NMDA receptors by prion proteins

Background: The precise physiological function of endogenous cellular prion protein (PrPC) remains unclear. It has been shown that PrP-null mice exhibit reduced LTP and greater susceptability to seizure mortality in several in vivo (e.g. kainic acid) models of epilepsy. In addition, PrP-null mice exhibit greater exctitotoxic cell death in response to kainic acid exposure. Methods: In our study we investigated the synaptic properties of WT and PrP-null mice. &#x0D; Results: Recordings in the CA1 layer of adult hippocampal slices showed that PrP-null mice exhibit a reduced threshold to evoked responses and no difference in paired-pulse facilitation relative to WT animals. In addition, greater excitability was observed in PrP-null slices in response to zero-Mg2+ induced seizure-like events. Recordings from mature hippocampal cultures showed slightly altered AMPA and GABAA miniature synaptic currents. NMDA mEPSCs were observed to be increased in amplitude and significantly prolonged in decay time. NMDA-evokved currents also exhibited markedly prolonged deactivation kinetics. This effect on evoked NMDA currents was reproduced in WT neurons by PrP-RNAi transfection, and eliminated by PrPC transfection into PrP-null neurons. &#x0D; Conclusions: These data suggest enhanced NMDA activity in PrP-null neurons. Consistent with this finding, in vitro and in vivo excitotoxicity assays demonstrated increased neuronal cell death in PrP-null cultures and animals upon transient exposure to NMDA. The prolonged deactivation kinetics were most consistent with functional activity/augmentation of NR2D NMDA receptor subunits, and PrP coimmunoprecipiated with NR2D NMDA receptor subunits. This enhanced NMDA receptor function was paralleleld by increased excitotoxicy in Prp-null mice. Our findings demonstrate a novel functional role for PrP as a modulator of synaptic NMDA currents and attributes a neuroprotective function to PrP.

Dentate Filter Function Is Altered in a Proepileptic Fashion during Aging

The elderly have an increased incidence and prevalence for seizure disorders. Further, since up to 50% of these cases have no identifiable antecedent, it has been hypothesized that aging of the central nervous system itself may be epileptogenic. Aged rats, compared to adults, exhibit a greater susceptibility to and severity of seizures associated with hippocampal activation. Whether this aging-related change reflects proconvulsive changes in limbic circuitry is unknown and thus was the focus of this study. Hippocampal slices from adult and aged Fischer 344 rats were examined using electrophysiological techniques. The dentate gyrus was our model region since it is involved with both wet-dog shakes and limbic seizures, and it is affected preferentially with age. No differences were noted between groups in field potential activity elicited with low frequency stimulation. In contrast, 5-Hz molecular layer stimulation could evoke multiple population spikes in approximately 40% of aged versus 0% of adult slices. Further, recording in CA3 revealed that this stimulation paradigm could elicit multiple spikes in aged, but not adult, slices that frequently evolved into spontaneous epileptiform bursts. This change in the capacity of the dentate to respond to and filter afferent input was associated with an aging-related decrease in the frequency of spontaneous IPSPs and an increased propensity for large amplitude prolonged EPSPs following disinhibition. These epileptogenic changes in dentate function and circuitry could contribute to the exacerbated susceptibility for hippocampal seizures in aged rodents, as well as the aging-related decline in spatial learning and memory.

Alzheimer Amyloid β‐Peptide A‐β25‐35 Blocks Adenylate Cyclase‐Mediated Forms of Hippocampal Long‐Term Potentiation

Progressive memory loss and deposition of amyloid beta (Abeta) peptides throughout cortical regions are hallmarks of Alzheimer's disease (AD). Several studies in mice and rats have shown that overexpression of amyloid precursor protein (APP) or pretreatment with Abeta peptide fragments results in the inhibition of hippocampal long-term potentiation (LTP) as well as impairments in learning and memory of hippocampal-dependent tasks. For these studies we have investigated the effects of the Abeta(25-35) peptide fragment on LTP induced by adenylate cyclase stimulation followed immediately by application of Mg(++)-free aCSF ("chemLTP"). Treatment of young adult slices with the Abeta(25-35) peptide had no significant effect on basal synaptic transmission in area CA1, but treatment with the peptide for 20 min before inducing chemLTP with isoproterenol (ISO; 1 microM) or forskolin (FSK;10 microM) + Mg(++)-free aCSF resulted in complete blockade of LTP. In contrast, normal ISO-chemLTP was observed after treatment with the control peptide Abeta(35-25). The ability of the Abeta(25-35) peptide fragment to block this and other forms of synaptic plasticity may help elucidate the mechanisms underlying hippocampal deficits observed in animal models of AD and/or AD individuals.

NMDA and non-NMDA receptors stimulation causes differential oxidative stress in rat cortical slices

Glutamate receptor activated neuronal cell death is attributed to a massive influx of Ca 2+ and subsequent formation of reactive oxygen species (ROS) but the relative contribution of NMDA and non-NMDA sub-types of glutamate receptors in excitotoxicity is not known. In the present study, we have examined the role of NMDA and non-NMDA receptors in glutamate-induced neuronal injury in cortical slices from young (20 ± 2 day) and adult (80 ± 5 day) rats. Treatment of slices with glutamate receptor agonists NMDA, AMPA and KA elicited the formation of reactive oxygen species (ROS) and neuronal cell death. In young slices, NMDA receptor stimulation caused a higher ROS formation and neurotoxicity, but KA was more effective in producing ROS and cell death in adult slices. AMPA exhibited an intermediate effect on ROS formation and toxicity in both the age groups. A significant protection in glutamate mediated ROS formation and neurotoxicity was observed in presence of NMDA or/and non-NMDA receptors antagonists APV and NBQX, respectively. This further confirms the involvement of both NMDA and non-NMDA receptors in glutamate mediated neurotoxicity. In adult slices, we did not find positive correlation between ligand induced neurotoxicity and mitochondrial depolarization. Though, NMDA and KA stimulation produced differential effect on ROS formation and neurotoxicity in young and adult slices, the mitochondrial depolarization was higher and comparable on NMDA stimulation in both the age groups as compared to KA, suggesting that the mitochondrial depolarization may not be a good indicator for neurotoxicity. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors are involved in glutamate mediated neurotoxicity but their relative contribution is highly dependent on the age of the animal.

Visually guided whole cell patch clamp of mouse supraoptic nucleus neurons in cultured and acute conditions

Recent advances in neuronal culturing techniques have supplied a new set of tools for studying neural tissue, providing effective means to study molecular aspects of regulatory elements in the supraoptic nucleus of the hypothalamus (SON). To combine molecular biology techniques with electrophysiological recording, we modified an organotypic culture protocol to permit transfection and whole cell patch-clamp recordings from SON cells. Neonatal mouse brain coronal sections containing the SON were dissected out, placed on a filter insert in culture medium, and incubated for at least 4 days to allow attachment to the insert. The SON was identifiable using gross anatomical landmarks, which remained intact throughout the culturing period. Immunohistochemical staining identified both vasopressinergic and oxytocinergic cells present in the cultures, typically appearing in well-defined clusters. Whole cell recordings from these cultures demonstrated that certain properties of the neonatal mouse SON were comparable to adult mouse magnocellular neurons. SON neurons in both neonatal cultures and acute adult slices showed similar sustained outward rectification above -60 mV and action potential broadening during evoked activity. Membrane potential, input resistance, and rapidly inactivating potassium current density (IA) were reduced in the cultures, whereas whole cell capacitance and spontaneous synaptic excitation were increased, perhaps reflecting developmental changes in cell physiology that warrant further study. The use of the outlined organotypic culturing procedures will allow the study of such electrophysiological properties of mouse SON using whole cell patch-clamp, in addition to various molecular, techniques that require longer incubation times.

Postnatal development of a new type of epileptiform activity in the rat hippocampus

Long-term application of Cs + (5 mM) induces an epileptiform field potential (Cs-FP) in area CA1 of the rat hippocampus, which is independent of N-methyl- d-aspartate (NMDA) and non-NMDA glutamate receptors and γ-aminobutyric acid (GABA) A receptors. To gain insight into possible mechanisms for the induction of the Cs-FP, we investigated the postnatal development of the response. In brain slices prepared from rats at different ages, the Cs-FP was evoked by stimulation of the Schaffer-collateral–commisural pathway. We found that expression of this potential was clearly dependent on the postnatal age. Thus, the Cs-FP was completely absent at 1 week of age. By 2 weeks, a reduced form of the response was observed, whereas slices taken from 3-week-old rats, displayed full Cs-FP, which were indistinguishable in size and shape from the adult form. In the presence of 4-aminopyridine, potentials resembling the Cs-FP were evoked. These potentials showed a similar age-dependency as the Cs-FP. The Na +/K + pump inhibitor dihydroouabain (DHO), when present during wash-in of Cs +, gave a partial block of the Cs-FP in adult slices. This effect was not seen when DHO was applied after development of the Cs-FP. The data indicate that the processes necessary for expression of the Cs-PF are absent at birth and develop during the second postnatal week. We propose that the Cs-FP depends on Cs + entry into presynaptic neurons, and that the Na +/K + pump contributes to this transport of Cs +. The observed age-dependency could therefore, in part, reflect the delayed development of the Na +/K + pump.

Synaptically-silent immature neurons show gaba and glutamate receptor-mediated currents in adult rat dentate gyrus.

The fate of adult-generated neurons in dentate gyrus is mainly determined early, before they receive synapses. In developing brain, classical neurotransmitters such as GABA and glutamate exert trophic effects before synaptogenesis. In order for this to occur in adult brain as well, immature non-contacted cells must express functional receptors to GABA and glutamate. In this investigation, patch-clamp recordings were used in adult rat dentate gyrus slices to assess the presence and analyze the characteristics of GABA- and glutamate-evoked currents in highly immature, synaptically-silent granule cells. Whole-cell patch-clamp recordings showed that all the analyzed cells responded to puff application of GABA and most of them responded to glutamate. Currents evoked by GABA were mediated exclusively by GABAA receptors and those elicited by glutamate were mediated by NMDA and AMPA/Kainate receptors. GABAA receptor-mediated currents were reduced by furosemide, which suggests that synaptically-silent immature neurons express high-affinity, alpha4-subunit-containing GABAA receptors. Gramicidin-perforated-patch recordings showed that GABAA receptor-mediated currents exerted a depolarizing effect due to high intracellular chloride concentration. Synaptically-silent immature cells shared morphological and electrophysiological properties with GFP-expressing, 7-day-old adult-generated granule layer cells, indicating that they could be in the first week of life, the period of maximal newborn cell death. Moreover, the presence of functional GABA and glutamate receptors was confirmed in these GFP-expressing cells. Present findings are mostly consistent with previous data obtained in female mice undergoing spontaneous activity and in transgenic mice, except for some inconsistencies about the presence of functional glutamatergic receptors. We speculate that adult-generated, non-contacted granule cells may be able to sense activity-related variations of GABA and glutamate extracellular levels. This condition is necessary, even if not sufficient, for these neurotransmitters to have a direct role in addressing cell survival.