Neurons Of Neonatal Rats Research Articles

P-379 - Preferential enhancement of inhibitory synaptic transmission by CS-722 in the ventral horn neurons of neonatal rats

P-379 - Preferential enhancement of inhibitory synaptic transmission by CS-722 in the ventral horn neurons of neonatal rats

Inhibition of lipid peroxidation attenuates axotomy-induced apoptotic degeneration of facial motor neurons in neonatal rats.

The purpose of this study was to investigate the role of oxygen radical-induced lipid peroxidative mechanisms in trophic deprivation-induced apoptotic motor neuronal degeneration by testing the ability of the 21-aminosteroid lipid peroxidation inhibitor tirilazad mesylate (U-74006F) to attenuate the retrograde degeneration of facial motor neurons following axotomy in 14-day-old rat pups. On day 0, the right facial nerve of each rat was transected at its point of exit from the stylomastoid foramen. Pups were treated orally with either 10 or 30 mg/kg U-74006F or cyclodextrin vehicle 10 min before axotomy, and post-treated once a day from days 1 to 6, and then once every other day from days 8 to 21. The rats were sacrificed 3 weeks post-transection and the surviving motor neurons, identified through choline acetyltransferase immunocytochemistry, were counted in three regions (planes) in the facial nucleus. In vehicle-treated rats, 56.2% (region A), 50.6% (region B), and 57.4% (region C) of the motor neurons in the ipsilateral facial nucleus survived 21 days following facial nerve axotomy in comparison to the non-axotomized contralateral nucleus (P < 0.0001). Treatment with 10 mg/kg U-74006F significantly enhanced motor neuron survival in regions B and C to 72.8% (P < 0.01) and 66.7% (P < 0.02%), respectively. The 30 mg/kg dose level also increased survival rates to 64.2% (P < 0.02) and 67.9% (P < 0.01), respectively. A second experiment demonstrated that oral dosing with U-74006F (30 mg/kg), when limited to the first 5 days after axotomy, also significantly blunted retrograde degeneration measured at 21 days post-axotomy. The efficacy of the lipid peroxidation inhibitor U-74006F in protecting a portion of the facial motor neuron pool from post-axotomy degeneration suggests that lipid peroxidation may play a mechanistic role in trophic deprivation-induced apoptotic neuronal death.

CNQX prevents spinal motor neuron death following sciatic nerve transection in newborn rats

A rapid and reproducible spinal motor neuron death occurs after sciatic nerve transection in neonatal rats. This neuronal death could be due to lack of retrogradely transported target derived neurotrophic factors, such as ciliary neurotrophic factor, brain-derived neurotrophic factor, leukemia inhibitory factor and glial cell line-derived neurotrophic factor. Another hypothesis suggests that glutamate and its receptors has been implicated as possible mechanism for motor neuron death. In order to investigate the effect of N-methyl- d-aspartate (NMDA) and non-NMDA receptor antagonists on axotomy-induced cell death in the spinal motor neurons of neonatal rats, we have studied neuroprotective effects of these receptor antagonists. Newborn rats were anesthetized with hypothermia. Sciatic nerve was transected near the obturator tendon in the left thigh. Animals were then treated daily with MK-801, APV, and CNQX for 14 days with intraperitoneal injections. Control animals received PBS in the same fashion. After the treatment, the number of spinal motor neurons in the L 4–6 was counted. MK-801 and APV did not show any significant neuroprotective effect. By contrast, the number of surviving motor neurons was greater in animals that were treated with 1.0, 2.0 and 4.0 mg/kg of CNQX. This neuroprotective effect was not dose-related. We demonstrate that neuroprotective effect of CNQX on axotomized motor neurons, raises a possibility that such a agent may have therapeutic potential in motor neuronopathy and amyotrophic lateral sclerosis.

Developmental changes in the electrophysiological properties of neonatal rat oculomotor neurons studied in vitro

The electrophysiological properties of oculomotor neurons were studied in neonatal rats aged 1–15 days. Action potentials were recorded from brainstem slices (frontal section) using the intracellular recording method at 35 °C. No significant age-dependent differences were detected in the resting potential (around −55 mV) and in the amplitude of the action potential (approximately 60 mV). However, the input resistance of oculomotor neurons declined with age from a mean of 60.8 MΩ for rats 1–3 days old to 17.0 MΩ for rats 14–15 days old. In addition, the duration of the action potential measured at the half-amplitude gradually decreased from 0.74 ms to 0.34 ms with increasing age. Increases were detected in the maximum rate of rise (from 117 V/s to 181 V/s) and the maximum rate of fall (from −67 V/s to −103 V/s) of the action potential. When long-lasting (500 ms) depolarizing current pulses were applied to oculomotor neurons, some neurons exhibited continuous repetitive discharge (i.e. tonic firing) while others showed transient discharge (phasic firing). The proportion of tonic-type neurons increased with age: the value was 9% for rats 1–5 days old, 37% for rats 6–10 days old and 54% for rats 11–15 days old. Concomitantly, the number of neurons showing a timedependent inward rectification increased and the average maximum frequency of the discharge rose from 150 to 420 Hz, approximately, with age. Furthermore, it was found that the electrophysiological properties of oculomotor neurons of rats 14–15 days old were similar to those in adult rats. It is concluded that oculomotor neurons in neonatal rats show rapid alterations in their electrophysiological properties and that the ratio of tonic-type to phasic-type neurons changes during the early stages of development.

Reverse transcriptase activity in human lymphocytes and brain tissue

Neonatal rats survive and avoid brain injury during periods of anoxia 25 times longer than adults. We hypothesized that oxygen activates and hypoxia suppresses NMDA receptor (NMDAR) responses in neonatal rat neurons, explaining the innate hypoxia tolerance of these cells. In CA1 neurons isolated from neonatal rat hippocampus (mean postnatal age [P] 5.8 days), hypoxia (PO2 10 mm Hg) reduced NMDA receptor-channel open-time percentage and NMDA-induced increase in [Ca2+]i (NMDA ΔCa2+) by 38 and 68% (P<0.01), respectively. In P20 neurons the reductions were not significant. In P3–10 CA1 neurons within intact hippocampal slices, hypoxia reduced NMDA ΔCa2+ by 52% (P=0.002) and decreased NMDA-induced death by 45% (P=0.004). Phalloidin, a microtubule stabilizer, prevented hypoxia-induced inhibition of NMDA ΔCa2+ in P3–10 neurons. To test whether NMDARs prevalent in neonates (NR1 plus NR2B or NR2D subunits) are inhibited by hypoxia compared with those in mature neurons (NR2A and NR2C), we expressed these receptors in Xenopus oocytes. Compared with responses in 21% O2, hypoxia (PO2 17 mm Hg) reduced currents from neonatal type NR1/NR2D receptors by 25%, increased currents from NR1/NR2C by 18%, and had no effect on NR1/NR2A or NR1/NR2B. Modulation of NMDARs by hypoxia may play an important role in the hypoxia tolerance of the mammalian neonate. In addition, oxygen sensing by NMDARs could play a significant role in postnatal brain development.

In vivo neurotrophic effects of GDNF on neonatal and adult facial motor neurons.

Motor neurons require neurotrophic factor(s) for their survival during development and for maintenance of function in adulthood. In vivo studies have shown that motor neurons respond to a variety of molecules, including ciliary neurotrophic factor, members of the neurotrophin family, and the insulin growth factor IGF-1 (refs 3-13). Here we investigate the potential motor neuron neurotrophic effects of glial-cell-line-derived neurotrophic factor (GDNF), initially identified as a neurotrophic factor for substantia nigra dopaminergic neurons. We find that GDNF is retrogradely transported, in a receptor-mediated fashion, by spinal cord motor neurons in neonatal rats. Local application of GDNF to the transected facial nerve prevents the massive motor neuron cell death and atrophy that normally follows axotomy in the neonatal period. In adult rats, GDNF administered locally or systemically can markedly attenuate the lesion-induced decrease of choline acetyltransferase immunoreactivity in the facial nucleus. Our data indicate that GDNF has very profound neurotrophic effects in vivo on developing as well as on adult motor neurons, and is the most potent motor neuron trophic factor found so far.

Inhibitory postsynaptic currents and the effects of GABA on visually identified sacral parasympathetic preganglionic neurons in neonatal rats

1. The actions of gamma-aminobutyric acid (GABA) on sacral parasympathetic preganglionic (SPP) neurons were examined in slice preparations using the whole cell patch-clamp technique. 2. Inhibitory postsynaptic currents (IPSCs), which were evoked by focal electrical stimulation, were recorded from SPP neurons in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a glutamate receptor antagonist. The IPSCs were substantially reduced by strychnine (1 microM), a glycine receptor antagonist. The remaining IPSCs were completely blocked by bicuculline (20 microM), a GABAA receptor antagonist. The mean peak amplitude of bicuculline-sensitive, GABAergic currents recorded at -60 mV was 53.6 +/- 10.9%, mean +/- SD (n = 8), of that of the total IPSCs. The GABAergic currents were reversed in polarity at about -30 mV, near the Cl- equilibrium potential. 3. GABA (5-50 microM) induced inward currents in SPP neurons with symmetrical internal and external Cl- concentrations. This response was completely blocked by 100 microM bicuculline. Muscimol (2-8 microM), a GABAA agonist, mimicked the GABA-induced responses, whereas a GABAB receptor agonist, baclofen (20-200 microM), produced responses in only a few cells. The GABA-induced currents reversed their polarity at approximately 0 mV, near the Cl- equilibrium potential. When the internal Cl- concentration was reduced, the reversal potential was shifted according to the Nernst equation for Cl-. 4. GABA-induced currents exhibited an outward "hump" between -35 and 15 mV. This voltage range coincided with that at which a depolarization-induced inward whole cell current was elicited.(ABSTRACT TRUNCATED AT 250 WORDS)

Perinatal cocaine exposure decreases the number of spontaneously active midbrain opamine neurons in neonatal rats

Perinatal cocaine exposure decreases the number of spontaneously active midbrain opamine neurons in neonatal rats

Postsynaptic Induction and Presynaptic Expression of Hippocampal Long-Term Depression

Long-term depression (LTD) is an activity-dependent decrease in synaptic efficacy that together with its counterpart, long-term potentiation, is thought to be an important cellular mechanism for learning and memory in the mammalian brain. The induction of LTD in hippocampal CA1 pyramidal neurons in neonatal rats is shown to depend on postsynaptic calcium ion entry through L-type voltage-gated calcium channels paired with the activation of metabotropic glutamate receptors. Although induced postsynaptically, LTD is due to a long-term decrease in transmitter release from presynaptic terminals. This suggests that LTD is likely to require the production of a retrograde messenger.

Neurotrophin 4/5 is a trophic factor for mammalian facial motor neurons.

The survival of developing motor neurons depends on factors secreted from skeletal muscles and from cells within the central nervous system. Although several members of the nerve growth factor protein family [neurotrophins (NTs)] are able to maintain developing rat motor neurons in vitro, only the brain-derived neurotrophic factor has been shown to have significant effects on the survival of motor neurons in vivo. In the present study, we demonstrate that NT-4/5 also prevents injury-induced death of facial motor neurons in neonatal rats. Furthermore, facial motor neurons express a functional receptor for NT-4/5, whereas mRNA-encoding NT-4/5 can be detected in their environment throughout embryonic and postnatal life. Thus, both NT-4/5 and brain-derived neurotrophic factor may be physiological survival factors for facial motor neurons and may serve as therapeutic agents for motor neuron disease.

Brain-derived neurotrophic factor rescues spinal motor neurons from axotomy-induced cell death.

Current ideas about the dependence of neurons on target-derived growth factors were formulated on the basis of experiments involving neurons with projections to the periphery. Nerve growth factor (NGF) and recently identified members of the NGF family of neuronal growth factors, known as neurotrophins, are thought to regulate survival of sympathetic and certain populations of sensory ganglion cells during development. Far less is known about factors that regulate the survival of spinal and cranial motor neurons, which also project to peripheral targets. NGF has not been shown to influence motor neuron survival, and whether the newly identified neurotrophins promote motor neuron survival is unknown. We show here that brain-derived neurotrophic factor (BDNF) is retrogradely transported by motor neurons in neonatal rats and that local application of BDNF to transected sciatic nerve prevents the massive death of motor neurons that normally follows axotomy in the neonatal period. These results show that BDNF has survival-promoting effects on motor neurons in vivo and suggest that BDNF may influence motor neuron survival during development.

Amiloride blocks glutamate-operated cationic channels and protects neurons in culture from glutamate-induced death

The diuretic amiloride has been suggested as a specific inhibitor of T-type neuronal Ca 2+ channels. The effects of amiloride on glutamate receptor-gated cationic channels and glutamate-induced, Ca 2+-dependent neuronal death were investigated in primary neuronal cultures from neonatal rats. In primary cultures of cerebellar granule neurons of the rat, receiving 50 μM glutamate for 15 min, at 22°C, in the absence of Mg 2+, about 80% of neurons were killed in about 24 hr. Exposure of neurons to such a pulse of glutamate, in the presence of various concentrations of amiloride, resulted in a dose-dependent protection from neurotoxicity (EC 50 300 μM, complete protection 1 mM). In voltage-clamped cortical and cerebellar neurons of neonatal rats in primary culture, 100 μM amiloride diminished (by about 25%) glutamate- and/or NMDA-evoked cationic currents, recorded in the whole-cell mode. About 80% of the NMDA- (20 μM) stimulated current was inhibited by 700 μM amiloride. The inhibitory effect of amiloride was not voltage-dependent. In outside-out membrane patches, excised from granule cells and held at −50 mV, 100 μM amiloride changed the NMDA-elicited single channel activity into a fast flickering between the open and closed states. The noise analysis of the data revealed that, although resembling the Mg 2+-induced flickering, the amiloride-induced channel block was more similar to the effects described for the action of local anaesthetics on the nicotinic cholinergic channel. The pharmacological relevance of this action of amiloride requires further characterization; the data point out the necessity of a cautious use of amiloride in studying neuronal function.

Evidence that lack of brain dopamine during development can increase the susceptibility for aggression and self-injurious behavior by influencing D 1-dopamine receptor function

Breese, George R., Hugh E. Criswell and Robert A. Mueller: Evidence That Lack of Brain Dopamine During Development Can Increase The Susceptibility for Aggression And Self-Injurious Behavior By Influencing D 1-Dopamine Receptor Function. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1990, 14 (Supple): S65–S80. 1. 1. Lesch-Nyhan disease has a defined neurological lesion that is accompanied by abnormal motor function, aggression and self-injurious behavior. 2. 2. The dopamine deficiency in Lesch-Nyhan disease has been modelled by destroying dopamine-containing neurons in neonatal rats with 6-hydroxydopamine. 3. 3. Because D 1-dopamine antagonists will block self-injurious behavior induced by L-DOPA in neonatal-6-OHDA-lesioned rats, D 1-dopamine antagonists are proposed as a potential therapy for aggression and self-injurious behavior in patients with these symptoms. 4. 4. The determination that the drug SCH-12679, which exhibited effectiveness against aggressiveness in mentally retarded patients, is a D 1-dopamine antagonist supports the view that new D 1-dopantine antagonists being developed will be an effective therapy for some types of aberrant behavior in this population.

Differences in the negative allosteric modulation of gamma-aminobutyric acid receptors elicited by 4'-chlorodiazepam and by a beta-carboline-3-carboxylate ester: a study with natural and reconstituted receptors.

Cl- currents elicited by gamma-aminobutyric acid (GABA) application were recorded with the whole-cell tight-seal technique from voltage-clamped cortical neurons of neonatal rats in primary culture. The peripheral benzodiazepine recognition site ligand 4'-chlorodiazepam [Ro 5-4864; 7-chloro-1,3-dihydro-1-methyl-5-(4-chlorophenyl)-2H-[1,4]-benzodiazep in-2- one] inhibited the GABA-generated currents in a dose-dependent manner. Also, a beta-carboline (DMCM; 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate methyl ester), acting as a negative allosteric modulator of GABAA receptors, reduced the intensity of GABA-generated currents with similar efficacy but greater potency. Flumazenil (Ro 15-1788; 8-fluro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-[1,5-a] [1,4]-benzodiazepine-3-carboxylate ethyl ester) antagonized DMCM inhibition but not that elicited by 4'-chlorodiazepam. The isoquinoline carboxamide PK 11195, an antagonist of 4'-chlorodiazepam effects in other systems, failed to antagonize the action of 4'-chlorodiazepam. The transient expression of various molecular forms of GABAA receptors in the human embryonic kidney cell line 293 allowed a study of the minimal structural requirements for the inhibition of GABA-induced Cl- currents by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. GABA-elicited Cl- currents in cells coexpressing alpha 1 and beta 1 subunits of GABAA receptors were inhibited by bicuculline and picrotoxin, but not by DMCM or 4'-chlorodiazepam. Conversely, the GABA currents in cells coexpressing alpha 1 beta 1 and gamma 2 subunits were inhibited by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. Since the Cl- currents generated by GABA in some molecular forms of GABAA receptors are inhibited by bicuculline and picrotoxin only, 4'-chlorodiazepam cannot be acting isosterically with picrotoxin.

Kinetic properties of single sodium channels in rat heart and rat brain.

Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics.

Neurosteroid pregnenolone sulfate antagonizes electrophysiological responses to GABA in neurons

Our earlier biochemical studies suggested that the neurosteroid pregnenolone sulfate (PS) may reduce γ-aminobutyric acid (GABA) action at the Cl − channel associated with GABA A receptors. In the present electrophysiological study the interaction of PS with the GABA A receptor was tested, using whole-cell voltage-clamp recordings from isolated cerebral cortical neurons of neonatal rats. At micromolar concentrations PS reversibly inhibited GABA-induced current, behaving as an allosteric receptor antagonist.

Phencyclidine and glycine modulate NMDA-activated high conductance cationic channels by acting at different sites

Glutamate activates high (40-50 pS) and low (5-15 pS) conductance cationic channels in outside-out patches excised from cultured cortical and cerebellar granule neurons of neonatal rats. In these neurons, the excitatory amino acid N-methyl-D-aspartic acid (NMDA) activates mainly high conductance channels. Phencyclidine (PCP) at 2 microM selectively reduces the number of NMDA-activated channel openings, at 20 microM it reduces the channel open-time. Glycine increases the opening frequency of high conductance NMDA-activated channels. This action is counteracted by PCP. This inhibition by PCP can be eliminated by reversing the polarity of the membrane patch. However, the effect of glycine is voltage independent. These results imply different sites of action for these two modulators.

Sprouting of striatal serotonin nerve terminals following selective lesions of nigro-striatal dopamine neurons in neonatal rat

The effects of neonatal intracisternal 6-hydroxydopamine (6-OHDA; 50 μg) treatment on striatal serotonin (5-HT) nerve terminals in rat have been characterized using histo- and neurochemical methods. The 6-OHDA lesion caused a 60% reduction of striatal dopamine (DA) concentration when analyzed in the adult stage, while 5-HT levels were increased by about 40% and 3H-5-HT uptake in vitro was increased by about 60%. Using computerized image analysis, a marked increase in 5-HT-like immunoreactive terminal density was found in both rostral (+200%) and caudal (+50%) striatum. Pretreatment with the DA uptake blocker amfolenic acid completely counteracted the 6-OHDA-induced alterations in both DA and 5-HT neurons in the striatum, while pretreatment with the noradrenaline uptake blocker desipramine had no significant effects. Regional analysis of 5-HT levels in the CNS after neonatal 6-OHDA treatment or the combined desipramine + 6-OHDA treatment showed no significant effect in any of the brain areas analyzed, apart from the observed 5-HT increase in striatum. It was furthermore observed that the striatal 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio was decreased, while the 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio was increased following the 6-OHDA lesion, indicating compensatory mechanisms in turnover of transmitters. These alterations were completely reversed after pretreatment with amfolenic acid. The present results support the view that the 5-HT hyperinnervation following neonatal 6-OHDA treatment is a collateral sprouting response induced by lesioning of the striatal DA neurons.

Reduction of [3H]substance P binding in the intermediolateral cell column after sympathectomy.

Guanethidine-induced destruction of sympathetic postganglionic neurons in neonatal rats leads to transneuronal degeneration of the sympathetic preganglionic neurons. Using this model, we have been able to show a approximately 35% decrease in [3H]substance P ([3H]SP) binding in the intermediolateral cell column--suggesting that sympathetic preganglionic neurons possess substance P receptors. Our results show that [3H]substance P binding in the intermediolateral cell column is dependent on the integrity of sympathetic postganglionic neurons.

Ionic and acid-base regulation of neurons and glia during seizures.

Much evidence shows that glia regulates the cation and anion content of brain interstitial space. In rats the pH and bicarbonate (HCO3-) concentration of neurons and glia were derived from carbon 14-labeled HCO3- and dimethyloxazolidinedione uptake into brain and cerebrospinal fluid. Acetazolamide increases the total CO2 concentration in neurons and decreases the pH and HCO3- concentration in glia. Inhibition of glial carbonic anhydrase (CA) reduces conversion of neuronally derived CO2 to HCO3-, glial pH is lowered, and neuronal CO2 accumulates. CA therefore has an essential role in regulating pH in neurons, glia, and interstitial fluid. In audiogenic seizure mice, glial CA activity is increased and glial anion transport is reduced. As the mice age, seizure susceptibility, the increased CA activity, and the defect in anion transport disappear concurrently. The enhanced CA activity in the glial cells of these mice is an adaptive mechanism to overcome the defect in anion transport that results from a deficiency of HCO3- -dependent and Na+- and K+ -dependent adenosine triphosphatase. Pentylenetetrazol stimulates neurons in neonatal rats, but after 10 days of age, when glia is present, it too is stimulated and the seizures are attenuated. Cobalt implantation in the cortex of rats also induces a glial response that ameliorates the focal seizures produced by this procedure.