Activating effect of substance P on nerve tissue in culture

The effect of substance P on explant development was investigated in organotypic cultures of rat sympathetic ganglia and spinal cord. The pattern of evolution, cellular composition, and dimensions of the growth zone were evaluated on the basis ofin vivo observations. It was found that this peptide exercises a significant growth-promoting effect at a concentrations of 10^–5–10^–12 M for sympathetic ganglia and 10^–5–10^–14 M for spinal cord culture. The growth zone of sympathetic ganglia measured 1.3–1.6 times the control level by the 14th day of culture at all effective concentrations. The area of outgrowth of spinal cord explants increased 2.0–5.2 fold by the sixth day of culture and peak response was recorded at concentrations of 10^–5 and 10^–12 M. This effect resembled response to opioid peptides [1, 3]. The likely physiological significance of regulatory peptides for the processes of nerve tissue development and regeneration is discussed in the light of these findings, together with the part played by the nociceptive/antinociceptive system in processes of histogenesis and repair.Institute of Experimental Cardiology, All-Union Cardiologic Research Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 610–615, September–October, 1986.     

Influence of opioid peptides on nerve tissue in vitro

The influence of opioid peptides (gamma- and beta-endorphins, leu- and met-enkephalins, as well as certain synthetic analogs of enkephalin) was investigated on organotypic cultures of rat spinal and sympathetic ganglia. The cellular composition and size of the zone of growth were evaluated on the basis of intravital observations and an analysis of the specimen obtained using the method of impregnation, according to Holmes and the detection of catecholamines with glyoxylic acid. It was established that under the action of all the investigated substances that possess high affinity for opiate receptors, growth of the neurites from an explant was enhanced, and the number of glial and fibroblastoid cells in the growth zone was increased. The effect was observed most distinctly on a model of sympathetic ganglia. The tested compounds exhibited a significant growth-stimulating effect in the range of concentrations 10^–8–10^–14 M. The maximum size of the growth zone of the explants of the sympathetic ganglia in the case of a mean effective concentration of the peptides 10^–10 M by the third to fifth day of culturing was approximately 2–2.5 times this value in the control. The reaction was similar to the response of the nerve cells to nerve growth factor, used as a standard. Thus, the opioid peptides exhibit a pronounced growth effect on the structures of the nerve tissue under conditions of culture. It is suggested that this group of compounds, together with its currently well-known functions, may play a definite role in processes of the development and regenera-of nerve tissue.Institute of Experimental Cardiology, All-Union Cardiologic Science Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 17, No. 4, pp. 550–557, July–August, 1985.     

T-588 Enhances Neurite Outgrowth and Choline Acetyltransferase in Cultured Rat Spinal Ventral Horn Neurons

T-588(R(-)-1-(benzo(b)thiophen-5yl)-2-[2(N,N-diethylamino)ethoxy]ethanol hydrochloride) is a novel compound which has been shown to exhibit a wide range of neurotrophic effects both in vivo and in vitro. This compound can slow the motor deterioration of wobbler mouse motor neuron disease. However, it is not known whether this compound has a trophic effect on spinal motor neurons. We have studied the effect of T-588 on neurite outgrowth and choline acetyltransferase(ChAT) activity in primary explant cultures of ventral spinal cord of fetal rats(VSCC). Cultures were treated with T-588 from day 1 to 1 week. T-588 treated VSCC, compared with control VSCC, had a significant neurite promoting effect at ranged between 10^–8 molar(M) and 10^–5 M, with 2.3 to 5.3 fold increased over that of control VSCC. In T-588 treated VSCC, ChAT activity was increased 1.5 times over that of control at 10^–6, and 10^–5 M respectively. Our data showing T-588 has neurotrophic action on VSCC and suggests a potential use of T-588 in treating diseases that involve degeneration and death of spinal motor neurons, such as motor neuropathy and motor neuron disease.     

Action of opioid peptides on nerve tissue growth and regeneration processes in the rat

Using organotypic cultures of the sympathetic ganglia and spinal cord from rats, studies have been made of the effect of opioid peptides on the development of the nervous tissue. It was found that endogenous opioid peptides (leu- and met-enkephalins, beta-endorphin) within the concentrations investigated (10(-9)-10(-10) M), stimulate the growth of neurites, affect the rate of migration and proliferation of the glial and fibroblast-like cells. The effect was observed at the 2nd--5th days of cultivation, depending on the object investigated. Naloxone, a blockator of the opiate receptors, does not abolish the stimulating effect of the opioid peptides. Using clonal line of fibroblast-like cells L6, it was shown that leu-enkephalin decreases the sensitivity to contact inhibition of growth. On the basis of the data obtained, it is suggested that endogenous opioid peptides act as non-specific factors of growth regulation in the development and regeneration of the nervous tissue. Taking into account the role of endorphins in the activity of noci-antinociceptive system possible significance of these compounds in post-injury reparation is discussed.     

Influence of Temocapril on Cultured Ventral Spinal Cord Neurons

Temocapril, a angiotensin-converting enzyme (ACE) inhibitor, was tested for neurotrophic activity in primary explant cultures of ventral spinal cord of fetal rats (VSCC). Temocapril had a remarkable effect on neurite outgrowth with a 4.2- to 5.1-fold increased over that of control VSCC at their effective concentrations. In temocapril-treated VSCC, choline acetyltransferase (ChAT) activity was also increased 2.4–3.2 times over that of control at 10^–9 and 10^–8 M, respectively. Our data suggest that temocapril is a candidate for neurotrophic factors on spinal motor neurons in vitro. A possible therapeutic role for temocapril in damaged motor neurons, such as in motor neuropathy and amyotrophic lateral sclerosis, remains to be defined.     

Peripheral Nerve Injury Modulates Neurotrophin Signaling in the Peripheral and Central Nervous System

Peripheral nerve injury disrupts the normal functions of sensory and motor neurons by damaging the integrity of axons and Schwann cells. In contrast to the central nervous system, the peripheral nervous system possesses a considerable capacity for regrowth, but regeneration is far from complete and functional recovery rarely returns to pre-injury levels. During development, the peripheral nervous system strongly depends upon trophic stimulation for neuronal differentiation, growth and maturation. The perhaps most important group of trophic substances in this context is the neurotrophins (NGF, BDNF, NT-3 and NT-4/5), which signal in a complex spatial and timely manner via the two structurally unrelated p75^NTR and tropomyosin receptor kinase (TrkA, Trk-B and Trk-C) receptors. Damage to the adult peripheral nerves induces cellular mechanisms resembling those active during development, resulting in a rapid and robust increase in the synthesis of neurotrophins in neurons and Schwann cells, guiding and supporting regeneration. Furthermore, the injury induces neurotrophin-mediated changes in the dorsal root ganglia and in the spinal cord, which affect the modulation of afferent sensory signaling and eventually may contribute to the development of neuropathic pain. The focus of this review is on the expression patterns of neurotrophins and their receptors in neurons and glial cells of the peripheral nervous system and the spinal cord. Furthermore, injury-induced changes of expression patterns and the functional consequences in relation to axonal growth and remyelination as well as to neuropathic pain development will be reviewed.     

Location of efferent sympathetic neurons and afferent neurons in spinal ganglia innervating the heart

Location and numbers of neurons associated with sympathetic innervation of the heart within the right stellate and accessory cervical ganglia, the spinal cord, and spinal ganglia were investigated using horseradish peroxidase retrograde axonal transport techniques in cats. The enzyme was applied to central sections of the anastomosis of the stellate ganglion with the vagus nerve, the inferior cardiac nerve, and the vagosympathetic trunk caudal to the anastomosis. Labeled neurons within the stellate ganglion were located close to the point of departure of the nerves and more thinly distributed in the accessory cervical ganglion. A group of labeled cells was found in the anastomosis itself. Preganglionic neurons associated with sympathetic innervation of the heat were detected at segmental levels T₁–T₅ in the spinal cord. Labeled neurons were diffusely located in the spinal ganglia, concentrated mainly at levels T₂–T₄.Medical Institute, Ministry of Public Health of the RSFSR, Yaroslavl'. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 106–111, January–February, 1989.     

Synaptosomal degradation of substance P and some other neuropeptides

Synaptosomes purified from spinal cord and from different rat brain areas exhibit peptide hydrolase activity, cleaving substance P (SP), bradykinin, THRH, LHRH, and neurotensin. The lowest activity for all the peptides tested was found in spinal cord, while the region with the highest degrading activity depended on the substrate: for substance P, it was striatum and cortex; for bradykinin, hypothalamus, and medulla oblongata; for THRH, striatum; for LHRH, midbrain; and for neurotensin, hippocampus. Degradation of substance P takes place at the plasma membrane of synaptosomes. Synaptosome ghosts cleave substance P (pH optimum 7–9,K _m–2.5×10^–5 M,V _max–130 nmol·hr^–1·mg protein^–1 and also a number of its C-terminal fragments. Effects of the inhibitors show that several different classes of peptidases and proteases are involved in the degradation process. Peptide cleavage represents the probable pathway of synaptosomal inactivation of substance P.     

Effect of millimeter band electromagnetic radiation on a culture of sensory neurons from the chick embryo

The effect of extra-high frequency electromagnetic radiation (EHF EMR) on the development of organotypical culture of the spinal ganglia of a 9–10 day-old chick embryo was investigated. EMR with a wavelength of 5.6 mm and a rate of flow density <1.0, 4.0, and >100 mW/cm² was used. The stimulating action of EMR at rate of flow density of 4.0 mW/cm², manifested in intensification of the growth of neurites of sensory neurons and the proliferation of the peripheral glia, was observed. EHF EMR with a density >100 mW/cm² exerted inhibitory influence. The possibility of using the stimulating effect of EHF EMR in medical practice for intensifying regeneration in pathology and after trauma of the peripheral nervous system is discussed.Neirofiziologiya/Neurophysiology, Vol. 25, No. 3, pp. 175–179, May–June, 1993.     

Regulation of Adenylyl Cyclase Signaling System in Cell Cultures of Infusoria Dileptus anser and Tetrahymena pyriformis by Peptides of Insulin Superfamily

Hormone-sensitive adenylyl cyclase signaling system (ACS) provides transduction of a wide spectrum of hormonal signals in cells of the higher eucaryotes. At the same time, ACS in the lower eucaryotes at present is practically not studied. We studied regulatory effects on ACS of the infusoria Dileptus anser and Tetrahymena pyriformis of peptide hormones of the higher eukaryotes—insulin, IGF-1, and relaxin, whose action on ACS of the higher eucaryotes was the subject of our earlier studies. The action of these hormones at concentrations of 10^–10–10^–8 M on the AC activity in infusoria had clearly stimulating character, the dose–effect curves being of a bell-shaped form with a maximum of the stimulating effect of the hormones at concentrations of 10^–9–10^–8 M. the shape of the curves and the value of the stimulating effect of the peptide hormones depended substantially on the level of the AC basal activity in homogenates of infusorian cell cultures. All the hormones (10^–8 M) stimulated GTP-binding activity of G-proteins. It was shown by the example of relaxin that its stimulating effect on GTP-binding in infusorian cells was dose-dependent and increased in the range of hormone concentrations from 10^–10 to 10^–8 M to reach its maximum at concentrations of 10^–8–10^–7 M. In the presence of suramin, an inhibitor of heterotrimeric G-proteins, the stimulating effects of the hormones on the GTP-binding and the AC activity decreased essentially or were absent completely. This indicates that the heterotrimeric G-proteins are ones of components of the signaling cascade that mediates regulatory effects of the hormones of the insulin group on the AC activity in infusorian cell cultures. Based on the obtained data, it is suggested that the basic molecular mechanisms of regulation of ACS by insulin and the related peptides that are similar to those found in the higher vertebrates already begin to be formed as early as at the level of the lower eucaryotes.     

Calnexin Deficiency Leads to Dysmyelination

Calnexin is a molecular chaperone and a component of the quality control of the secretory pathway. We have generated calnexin gene-deficient mice (cnx^−/−) and showed that calnexin deficiency leads to myelinopathy. Calnexin-deficient mice were viable with no discernible effects on other systems, including immune function, and instead they demonstrated dysmyelination as documented by reduced conductive velocity of nerve fibers and electron microscopy analysis of sciatic nerve and spinal cord. Myelin of the peripheral and central nervous systems of cnx^−/− mice was disorganized and decompacted. There were no abnormalities in neuronal growth, no loss of neuronal fibers, and no change in fictive locomotor pattern in the absence of calnexin. This work reveals a previously unrecognized and important function of calnexin in myelination and provides new insights into the mechanisms responsible for myelin diseases.     

Porphyrin synthesis in primary nervous tissue cultures from 10−3 M delta-aminolaevulinic acid in the presence of melatonin and neuropeptides

Of the primary neuronal tissue cultures (glia cell, neuronal cells, mixed and retina cultures), the neuronal cells of (cells + medium) display the highest total porphyrin production from 10^–3 M delta-aminolaevulinic acid (ALA). In the presence of 10^–3–10^–6 M melatonin, the quantity of total prophyrins produced by the neuronal cultures decreases in inverse proportion to the concentration. Oxytocin, lysine-vasopressin, CCK-8 sulphate ester and des-Tyr-gamma-endorphin in concentrations of 10^–5 and 10^–6 M block the porphyrin synthesis of the glia cells and display different effects on that of the neuronal cells. They enhance the total porphyrin synthesis of the cell cultures, with the exception of 10^–5 M des-Tyr-gamma-endorphin, which exerts an inhibitory effect on the glia cells.     

TNRNFLRFamide and SDRNFLRFamide modulate muscles of the stomatogastric system of the crab Cancer borealis

The effects of the extended FLRFamide-like peptides, TNRNFLRFamide and SDRNFLRFamide, were studied on the stomach musculature of the crab Cancer borealis. Peptide-induced modulation of nerve-evoked contractions was used to screen muscles. All but 2 of the 17 muscles tested were modulated by the peptides. In several muscles of the pyloric region, peptides induced long-lasting myogenic activity. In other muscles, the peptides increased the amplitude of nerve-evoked contractions, excitatory junctional potentials, and excitatory junctional currents, but produced no apparent change in the input resistance of the muscle fibers. The threshold concentration was 10^–10 M for TNRNFLRFamide and between 10^–9 M to 10^–8 M for SDRNFLRFamide. The absence of direct peptidecontaining innervation to these muscles and the wide-spread sensitivity of these muscles to the peptides suggest that TNRNFLRFamide and SDRNFLRFamide may be released from neurosecretory structures to modulate stomatogastric musculature hormonally. We speculate that hormonally released peptide will be crucial for maintaining appreciable muscle contraction in response to low-frequency and low-intensity motor discharge.Abbreviations cpv muscles cardiopyloric valve muscles - CG commissural ganglion - DG neuron dorsal gastric neuron - dgn dorsal gastric nerve - dvn dorsal ventricular nerve - EJC excitatory junctional current - EJP excitatory junctional potential - FaRPs FMRF-amide related peptides - gm muscles gastric mill muscles - lvn lateral ventricular nerve - mvn medial ventricular nerve - p muscles pyloric muscles - STG stomatogastric ganglion     

An analysis of dorsal root ganglia differentiation using three tissue culture systems

Summary The histogenesis of the dorsal root ganglia of chick embryos (ages 3 to 9 days) was followed in three different tissue culture systems. Organotypic explants included dorsal root ganglia connected to the lumbosacral segment of the spinal cord or isolated explants of the contralateral ganglia. Additionally, dissociated monolayer cultures of ganglia tissue were established. The gradual differentiation of progenitor neuroblasts into distinct populations of large ventrolateral and small dorsomedial neurons was observed in vivo and in vitro. Neurites developed after 3 days in the presence or absence of nerve growth factor in the medium. In contrast, autoradiographic analysis indicates that [³H]thymidine incorporation in neuronal cultures differed significantly from intact embryos. In vivo, the number of neuronal progenitor cells labeled with [³H]thymidine decreased in older embryos; in vitro, uptake of [³H]thymidine label was not observed in ganglionic progenitor cells regardless of the age of the donor embryo or the type of culture system. Lack of proliferation in ganglionic progenitor cells was not due to degeneration because vital staining and uptake of [³H]deoxyglucose indicated that neurons were metabolically active. Furthermore, the block in mitotic activity in vitro was limited to presumptive ganglionic neuronal cells. In the ependyma of the spinal cord segment connected to the dorsal root ganglia, neuronal progenitor cells were heavily labeled as were non-neuronal cells within both spinal cord and ganglia. Our results suggest that in vitro conditions can promote the differentiation of sensory neurons from early embryos (E3.5–4.5) without proliferation of progenitor cells.     

Interactions between prostaglandin E2 andd-ala2-met-enkephalinamide on adenylate cyclase activity in the guinea-pig superior cervical ganglion

Crude membrane fractions, obtained from superior cervical ganglia of normal and sympathectomized guinea-pigs, have been used to investigate the role of prostaglandin E₂ andd-ala²-met-enkephalinamide in the modulation of ganglionic adenylate cyclase as well as their functional interrelationship. In ganglia from normal animals the enzyme activity was stimulated and inhibited, respectively, by the prostaglandin (10^–4M) and by the opiate pentapeptide (10^–4M), while little or no effects were observed in denervated preparations. When the two substances were tested in combination, a supra-additive stimulation of adenylate cyclase activity was obtained both in normal and denervated ganglia. In the latter preparation the opiate increased prostaglandin E₂ specific binding, suggesting that the mechanism of supra-additivity could involve interactions at receptors level. Furthermore, the supra-additive stimulation of adenylate cyclase activity by the combination of the two drugs was obtained in a narrow range of concentrations since at low prostaglandin E₂ doses (10^–7–10^–6M) or at very high doses of the opiate (10^–3M), only the inhibitory effect ofd-ala²-met-enkephalinamide was evidenced.     

The distribution and turnover of tryptamine in the brain and spinal cord

Tryptamine levels have been determined in mouse brain regions and spinal cord and in rat spinal cord. They were; caudate nucleus 2.5 ng·g^–1, hypothalamus <0.5 ng·g^–1, hippocampus <0.7 ng·g^–1, olfactory bulb <0.7 ng·g^–1, olfactory tubercles <0.6 ng·g^–1, brain stem <0.4 ng·g^–1, cerebellum <1.0 ng·g^–1, and the rest 0.9 ng·g^–1. The mouse whole brain was found to have 0.5 ng·g^–1, the mouse spinal cord 0.3 ng·g^–1, and the rat spinal cord 0.3 ng·g^–1. These concentrations increased rapidly to 22.8 ng·g^–1, 14.2 ng·g^–1, and 6.6 ng·g^–1 respectively at 1 hr after 200 mg·kg^–1 pargyline. The turnover rates and half lives of tryptamine in the mouse brain and spinal cord and rat spinal cord were estimated to be 0.14 nmol·g^–1·h^–1 and 0.9 min; 0.054 nmol·g^–1·h^–1 and 1.5 min and 0.04 nmol·g^–1·h^–1 and 1.6 min respectively. The aromaticl-aminoacid decarboxylase inhibitors NSD 1034 and NSD 1055 reduced synthesis of tryptamine in controls and pargyline pretreated animals. Tryptophan increased the concentrations of mouse striatal tryptamine and 5-hydroxytryptamine and brain stem 5-hydroxyindole acetic acid.p-Chlorophenylalanine reduced formation of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid but did not change that of tryptamine.     

Is there a “dopaminergic glial cell”?

Intracellular cAMP increased 9-fold in cerebral hemisphere primary cultures after incubation with dopamine (10^–4M). The effect was dose- and time-dependent (10^–6 M-10^–4M; 2–10 minutes). It was mimicked, to some extent, by the partial agonist apomorphine (10^–5 M-10^–4 M) and antagonized by fluphenazine (10^–5 M-10^–4 M). The elevation of cAMP caused by dopamine was incompletely antagonized by propanolol (10^–5 M-10^–4 M), obviating an interaction with -adrenergic receptors. A -adrenergic effect was antagonized by propranolol but only slightly by fluphenazine. The effect of dopamine on cAMP-level was more pronounced in a subpopulation of the hemisphere culture, i.e. in astroglial cultures from the striatum, 12-fold compared with controls at 10^–4 M. No dopamine stimulated formation of cAMP was found in primary cultures from brain-stem. The results demonstrated some heterogeneity among astroglial cells. The cultures used contained mainly astroglial-like cells, as judged from immnohistochemical localization mainly astroglial-like cells, as judged from immunohistochemical localization of the glial specific proteins S 100 and GFA (-albumin). No mature neurons or oligodendroglial cells have so far been demonstrated in the cultures.     

Inhibitory effect of dopamine on the transmission of excitation in isolated rat spinal cord

The effects of dopamine on ventral root potential produced by a single supramaximal dorsal root stimulation of the dorsal root was investigated during experiments on isolated superfused spinal cord segments from 10–16-day old rats. A reciprocal dose-dependent inhibition of the mono- and polysynaptic components of reflex response was also observed. Minimum effective concentration was 1×10^–8 M dopamine. Extent of reflex response increased in step with dopamine concentration, so that the amplitude of the monosynaptic component of ventral root potential was decreased by 20% and 87% of baseline level by the action of 10^–4 and 10^–3 M dopamine respectively on the cord. The amplitude of the polysynaptic component was thereby decreased by an average of 18% and 87%. Findings indicate that dopaminergic brainstem-spinal pathways contribute to the governing of impulse transmission in the segmental reflex arcs. Inhibition of dopaminergic synaptic transmission probably underlies the increase in latency already described in the literature, as well as the increase observed in the threshold of reflex motor response to nociceptive action following either stimulation of the dopaminergic brainstem structures or intravenous administration of dopamine agonists.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 616–621, September–October, 1986.     

Segmental inhibitory response in the frog spinal cord during orthodromic motoneuron activation

Different types of reflex discharges were produced in various preparations by stimulating the dorsal root of isolated frog spinal cord. These ranged from multiphasic low-amplitude waves to distinctly synchronized monosynaptic response. The discharges were followed by facilitation in the former and deep, protracted inhibition of response to test dorsal root stimulation in the latter. When interstimulus intervals measured 40–50 msec, inhibitory action was less pronounced than at shorter (15–30 msec) or longer (60–100 msec) intervals, thus indicating that at least two types of inhibition were at work, one at an earlier and the other at a later stage. Strychnine at a concentration of 10^–5 M effectively reinforced the former and blocked the latter, while 10^–4 M d-tubocurarine attenuated both types of inhibition substantially. It is concluded that inhibition of response occurs mainly as a result of recurrent activation of inhibitory systems via recurrent motoneuron axon collaterals when frog spinal cord afferents are excited. Intensity of the later (presynaptic) and earlier (postsynaptic) inhibition of reflex transmission is determined by the degree of synchrony in motoneuronal discharge in response to orthodromic stimulation.Institute of Medical Radiology, Academy of Medical Sciences of the USSR, Obninsk, Kaluga Oblast. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 343–350, May–June, 1987.     

Effects of dopamine on background and evoked activity in interneurons of a spinal cord segment isolated from infant rats

The effects of 1·10^–5–1·10^–3 M dopamine on background and evoked interneuronal-activity was investigated during experiments on a spinal cord segment isolated from 11–18-day old infnat rats. Dopamine induced an increase in background firing activity rate in 52.5% and a reduced rate in 42.5% of the total sample of responding cells. Dopamine exerted a primarily inhibitory effect on interneuronal activity invoked by dorsal root stimulation, as witnessed by the reduced amplitude of the postsynaptic component of field potentials in the dorsal horn together with the fact that invoked activity was depressed in 66.7% of total interneurons responding to dopamine and facilitated in only 33.3% of these cells. All dopamine-induced effects were reversible and dose-dependent. Dopamine-induced effects disappeared after superfusing the brain with a solution containing 0–0.1 mM Ca²⁺ and 2 mM Mn²⁺, suggesting that this response is of transsynaptic origin. In other cells the excitatory or inhibitory action of dopamine also persisted in a medium blocking synaptic transmission; this would indicate the possibility of dopamine exerting depolarizing and hyperpolarizing effects on the interneuron membrane directly. Contrasting responses to dopamine in interneurons may be attributed to the presence of different types of dopamine receptors in the spinal cord.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 7–16, January–February, 1989.