Intracortical excitation of spiny neurons in layer 4 of cat striate cortex in vitro.

Recordings were made from pairs of neurons in cat striate visual cortex in vitro to study the AMPA-channel-mediated components of intracortical excitatory synaptic connections between layer 4 spiny neurons and between layer 6 and layer 4 spiny neurons. Forty-six of the 72 cells recorded were identified morphologically. They consisted of spiny stellate and pyramidal cells in layer 4, and pyramidal cells in layer 6. Connections between layer 4 excitatory cells involve excitatory postsynaptic potentials (EPSPs) averaging 949 microV, with an average coefficient of variation of 0.21 (n = 30). The synapses operate at very high release probabilities (0.69-0.98). With repetitive stimulation these EPSPs show varying degrees of depression, largely mediated by presynaptic changes in release probability. Four pairs of layer 4 cells were reciprocally connected. The connections from layer 6 to layer 4 involve smaller, more variable EPSPs, with an average amplitude of 214 microV, and average coefficient of variation 0.72 (n = 7). These synapses operate at moderately high release probabilities (0.37-0.56). They show facilitation with repetitive stimulation, mediated largely by presynaptic changes in release probability. One excitatory connection from a layer 4 neuron to a layer 6 pyramidal cell was also detected. Thus, layer 4 spiny neurons receive effective excitation from two intracortical sources that have different synaptic dynamics and are likely to contribute significantly to the temporal properties of these cells in vivo.

Synthesis and biological activity of substituted bis-(4-hydroxyphenyl)methanes as N-type calcium channel blockers.

Voltage activated calcium channel (VACC) blockers have been demonstrated to have utility in the treatment of stroke and pain. A series of aminomethyl substituted phenol derivatives has been identified with good functional activity and selectivity for N-type VACC's over sodium and potassium channels. The methods of synthesis and preliminary pharmacology are discussed herein.

Multiple parallel synthesis of N,N-dialkyldipeptidylamines as N-type calcium channel blockers.

Selective N-type Voltage Sensitive Calcium Channel (VSCC) blockers have shown utility in several models of stroke and pain. A series of N,N-dialkyldipeptidylamines with potent functional activity at N-type VSCC's has been identified. Multiple parallel synthesis of a focused array of thirty compounds using polymer-supported quenching reagents and preliminary pharmacology are presented. Eighteen compounds were identified with an IC50 below 1 microM in an in vitro functional assay.

Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas.

We describe the first potent and selective blocker of the class E Ca2+channel. SNX-482, a novel 41 amino acid peptide present in the venom of the African tarantula, Hysterocrates gigas, was identified through its ability to inhibit human class E Ca2+ channels stably expressed in a mammalian cell line. An IC50 of 15-30 nM was obtained for block of the class E Ca2+ channel, using either patch clamp electrophysiology or K+-evoked Ca2+ flux. At low nanomolar concentrations, SNX-482 also blocked a native resistant or R-type Ca2+ current in rat neurohypophyseal nerve terminals, but concentrations of 200-500 nM had no effect on R-type Ca2+ currents in several types of rat central neurons. The peptide has the sequence GVDKAGCRYMFGGCSVNDDCCPRLGCHSLFSYCAWDLTFSD-OH and is homologous to the spider peptides grammatoxin S1A and hanatoxin, both peptides with very different ion channel blocking selectivities. No effect of SNX-482 was observed on the following ion channel activities: Na+ or K+ currents in several cultured cell types (up to 500 nM); K+ current through cloned potassium channels Kv1.1 and Kv1. 4 expressed in Xenopus oocytes (up to 140 nM); Ca2+ flux through L- and T-type Ca2+ channels in an anterior pituitary cell line (GH3, up to 500 nM); and Ba2+ current through class A Ca2+ channels expressed in Xenopus oocytes (up to 280 nM). A weak effect was noted on Ca2+ current through cloned and stably expressed class B Ca2+ channels (IC50 > 500 nM). The unique selectivity of SNX-482 suggests its usefulness in studying the diversity, function, and pharmacology of class E and/or R-type Ca2+ channels.

Synaptic interactions between smooth and spiny neurones in layer 4 of cat visual cortex in vitro.

1. Dual intracellular recording was used to examine the interactions between neighbouring spiny (excitatory) and smooth (inhibitory) neurones in layer 4 of cat visual cortex in vitro. Synaptic connections were found in seventeen excitatory-inhibitory neurone pairs, along with one inhibitory-inhibitory connection. 2. Fast excitatory inputs onto smooth neurones (basket cells) from spiny cells (spiny stellate or pyramidal cells) (n = 6) produce large excitatory postsynaptic potentials (EPSPs) of up to 4 mV mean amplitude, whereas basket cells evoke slower inhibitory postsynaptic potentials (IPSPs) in their postsynaptic targets (n = 17), of smaller amplitude (up to 1.6 mV at membrane potentials of -60 mV). 3. Both types of PSP appear to be multiquantal, and both may exhibit depression of up to 60 % during short trains of presynaptic spikes. This depression can involve presynaptic and/or postsynaptic factors. 4. One-third (n = 5) of the spiny cell-smooth cell pairs tested were reciprocally connected, and in the one pair for which the suprathreshold interactions were comprehensively investigated, the pattern of basket cell firing was strongly influenced by the activity in the connected excitatory neurone. The basket cell was only effective in inhibiting spiny cell firing when the excitatory neurone was weakly driven.

Peripheral versus central potencies of N-type voltage-sensitive calcium channel blockers.

The ability of a series of synthetic analogues of omega-conopeptides MVIIA (SNX-111) and TVIA (SNX-185) to prevent electrically-evoked norepinephrine release from rat tail artery and hippocampal slice preparations was determined in an effort to identify voltage-sensitive calcium channel (VSCC) blockers that selectively target N-type VSCCs in central nervous system tissue. Electrical field stimulation (3 Hz, 1 ms in duration. 80 V for 1 min) caused a high and consistent tritium outflow from rat tail artery and hippocampal slice preparations preloaded with [3H]-norepinephrine. All conopeptides, chosen for their selective affinities for high-affinity SNX-111 binding sites (i.e., N-type VSCCs) over high-affinity omega-conopeptides MVIIC (SNX-230) binding sites (i.e., P/Q-type VSCCs), produced a concentration-dependent inhibition of calcium dependent electrically-evoked tritium outflow from both tail arteries and hippocampal slices: IC50s ranged from 1.2 nM to 1.2 microM. Blocking potencies (IC50s) in the tail artery assay were significantly correlated with those measured in the hippocampal slice preparation (r = 0.91, P = 0.00000012). There was a significant correlation between IC50s for blockade of hippocampal norepinephrine release and the inhibition of high-affinity [125I]-SNX-I11 binding in rat brain synaptosomes (r = 0.76, P = 0.00028). Blockade of hippocampal norepinephrine release was not significantly correlated with the inhibition of high-affinity SNX-230 binding (r = 0.46, P = 0.056). Maximum inhibition of tritium outflow in the tail artery assay was 22+/-1.4% of control, approximating the value (20.9+/-16.0% of control) obtained in the absence of extracellular Ca2+. In contrast, the maximum inhibition of tritium release from hippocampal slices was 36.8+/-2.5% of control (P < 0.05, compared to that of the tail artery assay). These results suggest that (1) N-type VSCCs alone mediate low frequency electrical stimulation-evoked neurotransmitter release from peripheral sympathetic efferents (tail artery) while both N-type and non-N type(s) mediate neurotransmitter release from CNS neurons (hippocampus); and (2) analogues of omega-conopeptides MVIIA and TVIA do not differentiate between N-type VSCCs mediating norepinephrine release from central and peripheral neural tissues.

Synthesis and structure-activity relationship of substituted 1,2,3,4-tetrahydroisoquinolines as N-type calcium channel blockers.

Voltage Activated Calcium Channel (VACC) blockers have been demonstrated to have utility in the treatment of pain and stroke. A series of aminomethyl substituted isoquinolinol derivatives with potent functional activity for N-type VACC's have been identified. Their synthesis and preliminary pharmacology are discussed herein.

Pharmacokinetics of SNX-111, a selective N-type calcium channel blocker, in rats and cynomolgus monkeys.

SNX-111, a selective N-type voltage-sensitive calcium channel blocker, is in clinical trials for the treatment of ischemia-induced brain injury and chronic pain. Pharmacokinetic studies were conducted in rats and cynomologus monkeys to determine the disposition of this compound when it is administered for 24 hr by continuous, constant-rate intravenous infusion. Venous blood samples for determination of SNX-111 plasma levels were collected at regular intervals immediately before, during, and after dosing. Plasma concentrations of SNX-111 equivalents were measured by radioimmunoassay. Pharmacokinetic parameters were derived from plasma SNX-111 concentration-time data using a two-compartment pharmacokinetic model. Results showed close correspondences between pharmacokinetic parameters determined for both species. There were no consistent gender- or dose-related differences in calculated kinetic parameters. In all cases, apparent steady-state plasma SNX-111 concentrations were achieved within 2-4 hr of initiating SNX-111 infusion. Steady-state volume of distribution values were approximately 40% of body weight, indicating extravascular dissemination of SNX-111 to both extracellular and intracellular fluids. Elimination curves contained two exponential components. The fast component (rat t1/2, alpha = 0.375 hr; monkey t1/2, alpha = 0.730 hr) accounted for approximately 97% of the unit impulse disposition function. The apparent terminal half-life ranged from 4.61 hr (rat) to 6.48 hr (monkey). Current findings constitute the first description of the pharmacokinetics of a member of the omega-conopeptide family of neuronal calcium channel blockers.

Excitatory synaptic inputs to spiny stellate cells in cat visual cortex.

In layer 4 of cat visual cortex, the monocular, concentric receptive fields of thalamic neurons, which relay retinal input to the cortex, are transformed into 'simple' cortical receptive fields that are binocular and selective for the precise orientation, direction of motion, and size of the visual stimulus. These properties are thought to arise from the pattern of connections from thalamic neurons, although anatomical studies show that most excitatory inputs to layer 4 simple cells are from recurrently connected circuits of cortical neurons. We examined single fibre inputs to spiny stellate neurons. We examined single fibre inputs to spiny stellate neurons in slices of cat visual cortex, and conclude that thalamocortical synapses are powerful and the responses they evoke are unusually invariant for central synapses. However, the responses to intracortical inputs, although less invariant, are strong enough to provide most of the excitation to simple cells in vivo. Our results suggest that the recurrent excitatory circuits of cortex may amplify the initial feedforward thalamic signal, subserving dynamic modifications of the functional properties of cortical neurons.

Structure-activity analysis of a Conus peptide blocker of N-type neuronal calcium channels.

The synthetic peptide SNX-111 corresponding to the sequence of the omega-conopeptide MVIIA from the venom of the marine snail Conus magus is a highly potent and selective antagonist of N-type calcium channels. We have synthesized and characterized a large number of analogs of SNX-111 in order to elucidate the structural features of the peptide involved in blocking N-type calcium channels. Comparison of the binding of SNX-111 and its analogs to rat brain synaptosomal membranes rich in N-type channels revealed that, among the four lysines and two arginines in the molecule, lysine in position 2 and arginines at position 10 and 21 are important for the interaction of SNX-111 with N-type channels. The importance of the middle segment from residues 9 through 14 for this binding interaction was revealed by substitution of the individual residues as well as by the construction of hybrid peptides in which the residues 9-12 in SNX-111 and another conopeptide, SNX-183, corresponding to a peptide SVIB from Conus striatus, were interchanged. Introduction of the sequence SRLM from SNX-111 in place of RKTS in position 9-12 in SNX-183 resulted in a 38-fold increase in affinity.

Differential effects of morphine on noxious stimulus-evoked fos-like immunoreactivity in subpopulations of spinoparabrachial neurons.

In previous studies we reported that although morphine dose dependently inhibits noxious stimulus-evoked expression of the c-fos proto-oncogene in the rat spinal cord, morphine was without effect in certain populations of presumed nociresponsive neurons, even under conditions of complete behavioral analgesia. To determine whether the neurons that continue to express the c-fos gene include projection neurons, we evaluated the effect of morphine on noxious stimulus-evoked c-fos expression in spinoparabrachial neurons retrogradely labeled with Fluoro-gold. In the formalin test, we found that morphine analgesia was associated with a significant reduction in the number of Fos-like-immunoreactive spinoparabrachial projection neurons in the lateral reticulated area of the neck of the dorsal horn. Morphine, however, did not reduce the number of Fos-like-immunoreactive spinoparabrachial projection neurons either in the superficial dorsal horn or in the area around the central canal. These results indicate that under conditions of morphine analgesia two distinct populations of spinoparabrachial neurons can be recognized on the basis of their expression of the c-fos gene in response to noxious stimulation. Since the expression of the c-fos gene has been correlated with neuronal activity, these data suggest that activity, and central transmission of nociceptive information, persists in certain nociresponsive projection neurons during morphine analgesia. Alternatively, if activity has, in fact, been blocked in these neurons, our results indicate that injury can produce significant molecular changes in neurons even though the neuronal activity and pain associated with the injury is blocked by morphine.

Characterization of the binding of omega-conopeptides to different classes of non-L-type neuronal calcium channels.

The interaction of two synthetic omega-conopeptides SNX-111 (MVIIA) and SNX-230 (MVIIC) both derived from the marine snail Conus magus, with non-L-type neuronal voltage-sensitive calcium channels (VSCC) in rat brain synaptosomal preparations has been investigated with the aid of well-characterized 125I derivatives of the two peptides. To assess the effects of iodination on the binding characteristics of SNX-111 and SNX-230, the corresponding peptides containing monoiodotyrosine in place of tyrosine, namely, SNX-259 ([127I]SNX-111) and SNX-260 ([127I]SNX-230), respectively, were prepared by solid-phase synthesis. Saturation analysis showed that [125I]SNX-111 and [125I]SNX-230 bound to two distinct classes of high-affinity sites with apparent Kd's of 9 and 11 pM and Bmax's of 0.54 and 2.2 pmol/mg protein, respectively. Kinetic analysis revealed that both peptides exhibited high association rates as well as rapid dissociation rates in contrast to the 125I derivative of the synthetic omega-conopeptide from Conus geographus, GVIA (SNX-124), which binds irreversibly to N-type channels in rat brain synaptosomes. Competition binding experiments with [125I]SNX-111 and [125I]SNX-124 established that both of them bind to the same site, namely, N-type VSCC. The site detected by the binding of [125I]SNX-230 is distinct from N-type VSCC since SNX-111 has very low affinity (K(i) = 135 nM) in competition studies. Recent findings that a novel high-voltage-activated calcium channel in rat cerebellar granule neurons is resistant to blockers of L-, N-, and P-type VSCC but is highly sensitive to SNX-230 suggest that the [125I]SNX-230 binding site may represent this novel type of calcium channel or another, as yet undescribed, VSCC.

Transneuronal spread of the pseudorabies virus after injection into the central nucleus of the amygdala in the rat.

The pseudorabies virus (PRV) is a swine alpha herpes virus that is widely used as a neural tracer because of its marked neurotropism and transneuronal transmissibility (Card et al., 1991, 1992; Strack and Loewy 1990). PRV has been used to retrogradely identify spinal cord and brainstem connections to various peripheral organs, but few anatomical studies have used CNS inoculation of PRV to investigate intrinsic brain connectivity. Improved knowledge of the mode and temporal pattern of transneuronal spread is essential for interpretation of PRV tracing studies, and is also a prerequisite to the use of this and other herpes viruses as vectors in the CNS. This study investigated the distribution of PRV labelling in the CNS at various time points after its injection into the central nucleus of the amygdala (CA). The results indicate that detection of PRV in a retrogradely labelled site at any given time after injection is not only a function of the number of synapses in the pathway from the injection site, but is also highly dependent on the axon lengths involved, much more than would be expected if fast axonal transport were the limiting factor. In addition, the window of time during which PRV may be detected in a given site is limited ultimately by neuronal destruction.

The optimal number of major histocompatibility complex molecules in an individual.

A straightforward argument is presented to calculate the number of different major histocompatibility complex (MHC) molecules in an individual that maximizes the probability of mounting immune responses against a large number of foreign peptides. It is assumed that increasing the number of MHC molecules per individual, n, has three different effects: (i) it increases the number of foreign peptides that can be presented; (ii) it increases the number of different T-cell receptors (TCRs) positively selected in the thymus; but (iii) it reduces the number of TCRs by negative selection. The mathematical analysis shows that n = 1/f maximizes the number of different TCRs that pass through positive and negative selection and that n = 2/f maximizes the probability to mount immune responses against a large fraction of foreign peptides. Here f is the fraction of TCRs deleted by one MHC molecule. Both results depend on approximations that are discussed in the paper. The model presented has implications for our understanding of the evolutionary forces acting on the MHC.