Rat Neocortical Tissue Research Articles

Differential inhibitory effects of drugs acting at the noradrenaline and 5‐hydroxytryptamine transporters in rat and human neocortical synaptosomes*

Although the amino acid sequences of rat and human 5-hydroxytryptamine (5-HT) and noradrenaline (NA) transporters (i.e. SERT and NET) are highly homologous, species differences exist in the inhibitory effects of drugs acting at these transporters. Therefore, comparison of the potencies of drugs acting at SERT and NET in native human and rat neocortex may serve to more accurately predict their clinical profile. Synaptosomes prepared from fresh human and rat neocortical tissues were used for [(3)H]-5-HT and [(3)H]-NA saturation and competition uptake experiments. The drugs tested included NA reuptake inhibitors (desipramine, atomoxetine and (S,S)-reboxetine), 5-HT reuptake blockers (citalopram, fluoxetine and fluvoxamine) and dual 5-HT/NA reuptake inhibitors (duloxetine and milnacipran). In saturation experiments on synaptosomal [(3)H]-5-HT and [(3)H]-NA uptake, the dissociation constants did not indicate species differences although a smaller density of both SERT and NET was observed in human tissues. In competition experiments with the various drugs, marked species differences in their potencies were observed, especially at SERT. The rank order of selectivity ratios (SERT/NET) in human neocortex was as follows: citalopram >or= duloxetine = fluvoxamine >or= fluoxetine > milnacipran > desipramine = atomoxetine > (S,S)-reboxetine. Significant species differences in these ratios were observed for duloxetine, atomoxetine and desipramine. This study provides the first compilation of drug potency at native human neocortical SERT and NET. The significant species differences (viz., human vs. rat) in drug potency suggest that the general use of rodent data should be limited to predict clinical efficacy or profile.

The effect of estrogen and progesterone on spreading depression in rat neocortical tissues

Although gender differences in the incidence of migraine with aura appear to be related to high circulating levels of ovarian hormones, the underlying mechanisms are not yet fully understood. Several studies have suggested a major role for spreading depression (SD) in the pathogenesis and symptomatology of migraine with aura. To investigate a possible role of SD in the association of high female hormones and attacks of migraine with aura, the effects of beta-estradiol and progesterone on SD were studied in rat neocortical tissues. Application of both hormones enhanced the repetition rate as well as the amplitude of SD in neocortical slices treated with hypotonic artificial cerebrospinal fluid. beta-Estradiol and progesterone also dose dependently increased the amplitude of SD induced by KCl microinjection. Both hormones exhibited a pronounced, persisting, and significant enhancement of long-term potentiation of the field excitatory postsynaptic potential in the neocortical tissues. The changes in SD characteristics in the presence of estrogen and progesterone may responsible for increased migraine with aura attacks associated by high female hormones. These hormones may exert their effects on SD via facilitation of synaptic transmission.

Differential sensitivity to induction of spreading depression by partial disinhibition in chronically epileptic human and rat as compared to native rat neocortical tissue

Spreading depression (SD) is characterized by a transient breakdown of neuronal function concomitant with a massive failure of ion homeostasis. It is a phenomenon that can be induced in neocortical tissue by raising excitability, e.g. injection of K +, application of glutamatergic agonists, or blocking Na +/K + ATPase. Here we report a novel method of SD induction using minimal disinhibition with application of low concentrations (5 μM) of the GABA A receptor blocker bicuculline. This procedure—while subthreshold for epileptiform activity—readily induced spontaneous SDs in native rat neocortical slices, accompanied by typical depolarizations of neurons and glial cells. In contrast, in human neocortical preparations obtained from epilepsy surgery, in ∼20% of the slices spontaneous epileptiform activity appeared with this bicuculline dosage without SDs. Raising the concentration of bicuculline to an epileptogenic dose (10 μM) in human tissue also resulted in the generation of epileptiform activity only. Likewise, in slices from pilocarpine-treated, chronically epileptic rats, bicuculline also only induced epileptiform activity without eliciting SDs. The experiments indicate that chronic epilepsy causes a differential sensitivity to partial GABA A receptor blockade with regard to induction of SD.

Preferential action of gabapentin and pregabalin at P/Q-type voltage-sensitive calcium channels: inhibition of K+-evoked [3H]-norepinephrine release from rat neocortical slices.

Gabapentin (GBP; Neurontin) and pregabalin (PGB; CI-1008), efficacious drugs in several neurological and psychiatric disorders, inhibit neurotransmitter release from mammalian brain slices at therapeutically relevant concentrations. A detailed investigation, exploring the basis for this in vitro phenomenon, focused on norepinephrine (NE) and rat neocortical tissue in complementary assays of neurotransmitter release and radioligand binding. The results are consistent with the hypothesis that GBP, PGB, and related substances decrease neocortical NE release by acting at the alpha2delta subunit of presynaptic P/Q-type voltage-sensitive Ca2+ channels (VSCC) subserving Ca2+ influx in noradrenergic terminals. The inhibitory action appears competitive with [Ca2+]o and preferential to those neurons undergoing prolonged depolarization. Other results indicate that the reduction of exocytotic NE release is independent of L- and N-type VSCC, classical drug/peptide binding sites on VSCC, Na+ channels, alpha2-adrenoceptors, NE transporter, and system L amino acid transporter. These findings suggest a selective modulation of P/Q-type VSCC that are implicated in neurotransmission and several GBP-responsive pathologies.

Different actions of general anesthetics on the firing patterns of neocortical neurons mediated by the GABA(A) receptor.

In cultured slice preparations of rat neocortical tissue, clinically relevant concentrations of volatile anesthetics mainly decreased action potential firing of neurons by enhancing gamma-aminobutyric acid (GABA(A)) receptor-mediated synaptic inhibition. The author's aim was to determine if other anesthetic agents are similarly effective in this model system and act via the same molecular mechanism. The actions of various general anesthetics on the firing patterns of neocortical neurons were investigated by extracellular single-unit recordings. Pentobarbital, propofol, ketamine, and ethanol inhibited spontaneous action potential firing in a concentration-dependent manner. The estimated median effective concentration (EC50) values were close to or below the EC50 values for general anesthesia. Bath application of the GABA(A) antagonist bicuculline (100 microM) decreased the effectiveness of propofol, ethanol, halothane, isoflurane, enflurane, and diazepam by more than 90%, indicating that these agents acted predominantly via the GABA(A) receptor. The depressant effects of pentobarbital and ketamine were not significantly reduced by bicuculline treatment. Drugs acting mainly via the GABA(A) receptor altered the firing patterns of neocortical cells in different manners. Diazepam reduced the discharge rates by decreasing the number of action potentials per burst, leaving the burst rate unaffected. In contrast, muscimol, GABA, propofol, and volatile anesthetics decreased the burst rate. Although several anesthetic agents acted nearly exclusively via the GABA(A) receptor, they changed the discharge patterns of cortical neurons in different ways. This finding is explained by GABA-mimetic or benzodiazepine-like molecular interactions.

Functional properties of rat and human neocortical voltage-sensitive sodium currents

1. The functional properties of sodium currents in acutely dissociated adult human, neonatal rat [postnatal day (P) 3 and P10], and mature rat (P21-23) neocortical pyramidal neurons were studied using whole-cell patch-clamp techniques. 2. The voltage dependence of activation and steady-state inactivation of neonatal rat sodium currents was shifted in the positive direction when compared with mature rat sodium currents. In contrast, no difference was detected between the voltage dependence of activation and steady-state inactivation of mature rat and adult human sodium currents. 3. The fast inactivation of rat (neonatal and mature) and human neocortical sodium currents were best fit with three components; a fast decay component, a slow decay component, and a persistent component. The magnitude of the persistent current in neocortical neurons averaged 1-3% of the peak current. Inactivation was faster for sodium currents in neonatal rat neocortical neurons than in mature neurons. No difference was detected in the kinetics of inactivation between mature rat and adult human sodium currents. 4. Saxitoxin (STX) inhibited neuronal sodium currents at nanomolar concentrations in neonatal and mature rat and adult human neocortical neurons. STX-insensitive channels were not detected. 5. STX affinity was also assayed using 3H-STX. A single high-affinity binding site was found in neonatal rat, mature rat, and adult human neocortical tissue. A developmental increase in STX binding site density in the rat neocortex was tightly correlated with the increase in the sodium current density (normalized to cell capacitance). Human neocortical tissue and mature rat neocortical tissue did not differ in STX binding site density or sodium current density. 6. From these electrophysiological and autoradiographic studies we conclude that 1) the increase in the normalized sodium current density and STX binding density with age postnatally reflects an increase in binding sites of sodium channels functionally expressed on neuronal membranes, 2) the functional differences in channel behavior with maturation can explain the higher threshold for excitation in neonatal neocortical neurons and the increase in accommodation or adaptation in firing in the mature neuron, and 3) mature rat neocortical neurons represent a valid model for the study of adult human pyramidal neocortical neurons in terms of Na+ channel expression and function.

Dissociation of neurite-promoting activity and protease-inhibiting function of a2-macroglobulin in culture

The relationship between the neurite-promoting effect and the protease-inhibiting function of a 2-macroglobulin ( a 2M) was examined in a culture of dissociated neurons from embryonic rat neocortical tissue. The neurite-promoting effect of various protease inhibitors (soybean trypsin inhibitor, a 1-antitrypsin, aprotinin, phenylmethanesulfonyl fluoride, leupeptin, antipain, human a 2M and purified rat a 2M) was investigated. At lower concentrations, only a 2M significantly promoted neurite outgrowth. Furthermore, a 2M-trypsin complexes, which had lost their protease-inhibiting function and exposed their receptor-recognition site, promoted neurite outgrowth more efficiently than native a 2M. These results indicate the possibility that the neurite-promoting effect of a 2M is mediated by its receptor-binding activity but is not directly related to its protease-inhibiting function.

Local connections in transplanted and normal cerebral cortex of rats.

Injections of the fluorescent tracer Fluoro-Gold were made in transplanted and normal cerebral cortex of rats in order to investigate and compare the local connectivities of both. In the normal somatosensory cortex, small injections in superficial layers (I to III) produced retrograde cell labeling below the injection site in two bands: in layer V and in the deep part of layer VI. Pieces of embryonic rat neocortical tissue were transplanted into a cavity made in the somatosensory cortex of young adult rats. After a survival period of 2-3 months, small injections of Fluoro-Gold were made in the superficial part of the grafts. These injections revealed multiple clusters of intratransplant-projecting cells. No callosal or thalamic neurons were labeled in these experiments. On occasion, a bilaminated pattern of retrograde cell labeling was observed inside the transplants. In both transplanted and normal cortices, pyramidal and non-pyramidal cells were retrograde-labeled. We conclude that in the neocortical transplants there is a pattern of local connectivity that is reminiscent of the pattern of intracortical connectivity in the normal neocortex in at least two aspects: first, the retrograde-labeled cells tended to form clusters or bands; second, both pyramidal and non-pyramidal cells were labeled.

Interaction between adenosine generated endogenously in neocortical tissues, and homocysteine and its thiolactone

[ 14C]Adenine derivatives in normal guinea pig or rat neocortical tissues maintained by superfusion included ATP, ADP and AMP collectively forming some 98% of the acid-extracted 14C; adenosine, inosine and hypoxanthine each at less than 0.5% and S-adenosylhomocysteine at about 0.1%. l-Homocysteine and/or its thiolactone increased only a little the S-adenosylhomocysteine. The superfusion fluid carried from the tissue per minute about 0.1% of its acid-extractable [ 14C]adenine derivatives. Electrical stimulation of the superfused tissue increased 10-fold its output of [ 14C]adenine derivatives and diminished the 5′-nucleotides in the tissue to 94% of the acid-extractable [ 14C]adenine derivatives, the remainder being adenosine, inosine and hypoxanthine with little change in S-adenosylhomocysteine. Homocysteine in the superfusion fluids now caused large increases in tissue S-adenosylhomocysteine, which became the preponderant non-nucleotide 14C-derivative when homocysteine was 0.1 mM or greater. The total [ 14C]adenine conversion to non-nucleotide derivatives then increased and the 5′-nucleotides fell to 88% of the total. It is concluded that concentration relationships observed in the action of homocysteine make it feasible that convulsive conditions and mental changes associated with administered homocysteine and with homocystinuria are due to cerebral adenosine concentrations being diminished through formation of S-adenosylhomocysteine. Adenosine is preponderantly depressant in cerebral actions; effects of the S-adenosylhomocysteine produced may also be relevant.

EVIDENCE FOR THE PHARMACOLOGICAL SIMILARITY BETWEEN THE CENTRAL PRESYNAPTIC MUSCARINIC AUTORECEPTOR AND POSTSYNAPTIC MUSCARINIC RECEPTORS

Twenty antagonist substances with varying potencies for central and peripheral postsynaptic muscarinic receptors have been examined for effects on the central presynaptic muscarinic autoreceptor. This has been monitored by measuring the stimulating effects of the substances on acetylcholine synthesis by rat neocortical tissue prisms. Dose-response curves for selected agents showed that maximal stimulation of synthesis was to 136-140% of the value without an antagonist. At a concentration of 1 microM, 17 of the substances caused a significant increase in synthesis, whilst at 0.01 microM significant stimulation occurred with only atropine, dexetimide, N-methyl-piperdin-4-yl (R)-2-cyclohexyl-2-hydroxyl-2-phenylacetate, quinuclidinyl benzilate (QNB) and scopolamine. Linear regression analysis between synthesis values obtained with the substances and published data for the effects on either cholinoceptor-agonist induced contraction of guinea-pig ileum or the binding of [3H]-QNB to rat forebrain membranes gave correlation coefficients of r = 0.84 (P less than 0.01), and r = 0.75 (P less than 0.02) respectively. The results provide no indication of a pharmacological difference between the central presynaptic muscarinic autoreceptor and central and peripheral postsynaptic muscarinic receptors.