Nesfatin-1 exerts long-term effect on food intake and body temperature.

OBJECTIVE: To determine whether the anorexigenic peptide, nesfatin-1 affects energy expenditure, and to follow the time course of its effects. DESIGN: Food intake duration, core body temperature, locomotor activity and heart rate of rats were measured by telemetry for 48 h after a single intracerebroventricular injection of 25 or 100 pmol nesfatin-1 applied in the dark or the light phase of the day. Body weight, food and water intake changes were measured daily. Furthermore, cold-responsive nesfatin-1/NUCB2 neurons were mapped in the brain. RESULTS: Nesfatin-1 reduced duration of nocturnal food intake for 2 days independently of circadian time injected, and raised body temperature immediately, or with little delay depending on the dose and circadian time applied. The body temperature remained higher during the next light phases of the 48 h observation period, and the circadian curve of temperature flattened. After light phase application, the heart rate was elevated transiently. Locomotion did not change. Daily food and water intake, as well as body weight measurements point to a potential decrease in all parameters on the first day and some degree of compensation on the second day. Cold-activated (Fos positive) nesfatin-1/NUCB2 neurones have been revealed in several brain nuclei involved in cold adaptation. Nesfatin-1 co-localised with prepro-thyrotropin-releasing hormone in cold responsive neurones of the hypothalamic paraventricular nucleus, and in neurones of the nucleus raphe pallidus and obscurus that are premotor neurones regulating brown adipose tissue thermogenesis and skin blood flow. CONCLUSION: Nesfatin-1 has a remarkably prolonged effect on food intake and body temperature. Time course of nesfatin-1's effects may be varied depending on the time applied. Many of the nesfatin-1/NUCB2 neurones are cold sensitive, and are positioned in key centres of thermoregulation. Nesfatin-1 regulates energy expenditure a far more potent way than it was recognised before making it a preferable candidate anti-obesity drug.

SARS-CoV-2 entry sites are present in all structural elements of the human glossopharyngeal and vagal nerves: clinical implications.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infections result in the temporary loss of smell and taste (anosmia and dysgeusia) in about one third of confirmed cases. Several investigators have reported that the viral spike protein receptor is present in olfactory neurons. However, no study has been published to date showing the presence of viral entry sites angiotensin-converting enzyme 2 (ACE2), neuropilin1 (NRP1), and TMPRSS2, the serine protease necessary for priming the viral proteins, in human nerves that are responsible for taste sensation (cranial nerves: VII, IX and X). We used immunocytochemistry to examine three postmortem donor samples of the IXth (glossopharyngeal) and Xth (vagal) cranial nerves where they leave/join the medulla from three donors to confirm the presence of ACE2, NRP1 and TMPRSS2. Two samples were paraffin embedded; one was a frozen sample. In addition to staining sections from the latter, we isolated RNA from it, made cDNA, and performed PCR to confirm the presence of the mRNAs that encode the proteins visualized. All three of the proteins required for SARS-CoV-2 infections appear to be present in the human IXth and Xth nerves near the medulla. Direct infection of these nerves by the COVID-19 virus is likely to cause the loss of taste experienced by many patients. In addition, potential viral spread through these nerves into the adjacent brainstem respiratory centers might also aggravate the respiratory problems patients are experiencing.

Tuberoinfundibular peptide of 39 residues- immunoreactive fibers in the zona incerta and the supraoptic decussations terminate in the neuroendocrine hypothalamus.

Tuberoinfundibular peptide of 39 residues (TIP39) is expressed by neurons in the subparafascicular area, the posterior intralaminar complex of the thalamus and the pontine medial paralemniscal nucleus. TIP39-positive fibers from these areas do not form individual bundles or fascicles, they join other pathways to reach their innervated brain areas. Fibers arise from TIP39 perikarya located in the subparafascicular area and the posterior intralaminar complex of the thalamus could be followed to the hypothalamus. After uni- and bilateral posterolateral surgical deafferentations of the hypothalamus, accumulation of TIP39 immunoreactivity was observed in the fibers caudal to the knife cut, while it disappeared completely rostral to the transection. In serial sections of the forebrain, we could follow TIP39-ir fibers coursing within the zona incerta and the supraoptic decussations. TIP39-positive fibers that join the incerto-hypothalamic pathway reach the medio-dorsal part of the hypothalamus and form moderate to high density networks in the dorsomedial and paraventricular nuclei. The other set of TIP39-positive axons from the subthalamic area join the fibers of the supraoptic decussations and run in an antero-medial direction through the most ventral portion of the hypothalamus up to the retrochiasmatic area, where they crossover. A certain portion of these TIP39-positive fibers terminates in the territories of the arcuate and the medial preoptic nuclei, as well as in the retrochiasmatic area.

Neuronal activation in the CNS during different forms of acute renal failure in rats.

The effect of experimentally induced acute renal failure (ARF) on neuronal cell activation was investigated by immunohistochemistry for Fos and Fra-2 in the rat brain. ARF in rats was induced by bilateral nephrectomy (BNX), bilateral ureter ligature (BUL) and uranyl acetate injection with proper controls (sham-operation or saline injections, respectively). To follow the effect of the development of ARF, rats were killed 30 and 60 min, and 3, 12, 24 and 72 h after surgery, or 3 h to 12 days after uranyl acetate injections. In the BUL and BNX rats, urea and creatinine rose markedly in the plasma within 72 h, while in the uranyl acetate-injected rats the highest levels were observed on the 7th day, followed by a marked decline. At each time-point of the three different, experimentally induced ARF, the presence of Fos- and/or Fra-2-immunoreactive neurons was determined in 120 different brain areas and nuclei. In general, the 73 of 120 brain areas that showed time and intensity dependent activation in response to ARF can be classified into four groups: 1) biogenic amine (noradrenaline, adrenaline, histamine and 5-HT) expressing cell groups in the lower brainstem, 2) "stress-sensitive" forebrain areas, with regard to certain hypothalamic, limbic and cortical areas, 3) neuronal cell groups that participate in the central regulation of body and brain water and electrolyte homeostasis, including the circumventricular organs, and 4) central autonomic cell groups, especially visceral sensory cell groups in the brain, which are in primary, secondary or tertiary connections with renal afferents. Data presented here indicate that a wide variety of neurons in several regulatory mechanisms is affected by ARF-induced peripheral and central alterations.

Localization of targets for anti-ulcer drugs in cells of the immune system.

The gastric mucosa consists of the epithelium, which lines the lumen, the lamina propria, and the muscularis mucosae. The targets of drugs used to treat stomach and duodenal ulcers are thought to be the acid-secreting parietal cells of the epithelium. However, immune cells in the lamina propria are the only cells that showed detectable messenger RNAs for histamine, muscarinic, gastrin, and dopamine receptors by in situ hybridization histochemistry. None of the epithelial cells expressed any of these messenger RNAs. Thus, the targets of antiulcer drugs seem to be cells of the immune system in the gut and not parietal cells, as generally believed. This conclusion may revise the thinking about ulcer formation and may shed light on the etiology of such chronic small intestinal diseases as Crohn's disease.

Thyrotropin-releasing hormone in specific nuclei of rat brain.

The regional distribution of thyrotropin-releasing hormone (TRH) in rat brain was studied. The greatest concentration of TRH was found in the median eminence. High concentrations were also found in several hypothalamic nuclei. Outside the hypothalamus, relatively large amounts of TRH were found in the septal and preoptic areas.

Induced expression of the glucocorticoid receptor in the rat intermediate pituitary lobe.

Synthesis and release of pro-opiomelanocortin-derived peptides are under differential regulation in the anterior and intermediate lobes of the pituitary. Glucocorticoids inhibit synthesis of pro-opiomelanocortin-related peptides in the anterior lobe but not in the intermediate lobe. These two lobes are also characterized by differences in neural innervation and blood flow, both of which may represent routes of access for regulatory factors (the intermediate lobe is avascular). Immunoreactive glucocorticoid receptor, which can be demonstrated in many tissues, is absent from the intermediate lobe. Immunocytochemistry was used to demonstrate the presence of immunoreactive glucocorticoid receptor in the intermediate lobe after pituitary stalk transection, neurointermediate lobe grafts to kidney capsule, or monolayer culture of neurointermediate pituitary cells. This appearance of the glucocorticoid receptor is presumably a consequence of removal of intermediate pituitary cells from neural influences that may be responsible for inhibiting their expression under normal conditions in vivo.

Beta-adrenergic mechanism of insulin-induced adrenocorticotropin release from the anterior pituitary.

Intraperitoneal administration of insulin to control rats and to rats with pituitary stalk transections or with lesions of the median eminence resulted in increased plasma adrenocorticotropin (ACTH) levels. The insulin-induced stimulation of ACTH release was blocked in both the control and lesioned animals by prior treatment with either the beta-adrenergic antagonist propranolol or the glucocorticoid analog dexamethasone. The direct application of insulin to primary cultures of the anterior pituitary did not evoke ACTH release or affect the maximal ability of corticotropin-releasing factor or epinephrine to stimulate ACTH secretion. The results suggest that insulin stimulates ACTH release by a mechanism in which catecholamines of peripheral origin act directly on the anterior pituitary.

Methionine and leucine enkephalin in rat neurohypophysis: different responses to osmotic stimuli and T2 toxin.

Specific radioimmunoassays were used to measure the effects of hypertonic saline (salt loading), water deprivation, and trichothecene mycotoxin (T2 toxin) on the content of methionine enkephalin (ME), leucine enkephalin (LE), alpha-neoendorphin, dynorphin A, dynorphin B, vasopressin, and oxytocin in the rat posterior pituitary. Concentrations of vasopressin and oxytocin decreased in response to both osmotic stimuli and treatment with T2 toxin, but the decrease was greater with osmotic stimulations. Similarly, concentrations of LE and dynorphin-related peptides declined after salt loading and water deprivation; LE concentrations also decreased after treatment with T2 toxin. The concentration of ME decreased after water deprivation, did not change after salt loading, and increased after T2 toxin treatment. The differentiating effects of these stimuli on the content of immunoreactive LE and ME are consistent with the hypothesis that LE and ME may be localized in separate populations of nerve endings with different roles in the posterior pituitary.

Stressor specificity of central neuroendocrine responses: implications for stress-related disorders.

Despite the fact that many research articles have been written about stress and stress-related diseases, no scientifically accepted definition of stress exists. Selye introduced and popularized stress as a medical and scientific idea. He did not deny the existence of stressor-specific response patterns; however, he emphasized that such responses did not constitute stress, only the shared nonspecific component. In this review we focus mainly on the similarities and differences between the neuroendocrine responses (especially the sympathoadrenal and the sympathoneuronal systems and the hypothalamo-pituitary-adrenocortical axis) among various stressors and a strategy for testing Selye's doctrine of nonspecificity. In our experiments, we used five different stressors: immobilization, hemorrhage, cold exposure, pain, or hypoglycemia. With the exception of immobilization stress, these stressors also differed in their intensities. Our results showed marked heterogeneity of neuroendocrine responses to various stressors and that each stressor has a neurochemical "signature." By examining changes of Fos immunoreactivity in various brain regions upon exposure to different stressors, we also attempted to map central stressor-specific neuroendocrine pathways. We believe the existence of stressor-specific pathways and circuits is a clear step forward in the study of the pathogenesis of stress-related disorders and their proper treatment. Finally, we define stress as a state of threatened homeostasis (physical or perceived treat to homeostasis). During stress, an adaptive compensatory specific response of the organism is activated to sustain homeostasis. The adaptive response reflects the activation of specific central circuits and is genetically and constitutionally programmed and constantly modulated by environmental factors.

Direct neuronal projection from a brainstem thermosensitive cell group to the preoptic thermoregulatory center.

The preoptic area orchestrates thermoregulatory responses in homeotherm animals and humans. This thermoregulatory center receives thermal information about core body and skin temperatures, and in turn, it induces thermogenic responses. The physiology of effector mechanisms has been described in detail outlining the brain areas participating in the execution of thermal responses. Previous studies have presented evidence of peripheral thermosensation, existence of skin thermoreceptors, participation of spinal and brainstem sensory neurons in thermal stress, but only recently has been identified the first evidence of an ascending neuronal pathway transmitting thermal signal to the preoptic thermoregulatory center. Nevertheless, a few brainstem areas have not been linked to an afferent or efferent thermal pathway and the neuronal network of thermoafferent signals has only partially been identified. In the present study, we identified a distinct ascending neuronal projection that originates from the thermoreactive cells of the peritrigeminal nucleus in the medulla oblongata, and projects to the thermoreactive cells of the medial preoptic area in the hypothalamus of rats. First, we have demonstrated retrogradely labeled thermoreactive neurons in the parabrachial, pontine and peritrigeminal cells following the injection of pseudorabies virus, a retrograde multi-synaptic tract tracer, into the ventrolateral subdivision of the medial preoptic area. Confirming the existence of a direct neuronal connection, we detected biotinylated dextran amine (BDA) containing axonal fibers and boutons around thermoreactive cells of the ventrolateral subdivision of the medial preoptic area after BDA injection into the peritrigeminal nucleus that is known to respond the temperature changes. Our findings indicate the existence of a so far unrecognized ascending direct neuronal pathway that transmits thermal signal from the lower brainstem to the thermoregulatory preoptic center.

Gender-related urocortin 1 and brain-derived neurotrophic factor expression in the adult human midbrain of suicide victims with major depression.

In postmortem brains of patients with major depression, the expression of corticotrophin-releasing factor (CRF) is enhanced and that of brain-derived neurotrophic factor (BDNF) decreased. In mice over-expressing neuronal CRF (an animal model for depression) the expression of urocortin 1 (Ucn1) in the non-preganglionic Edinger-Westphal nucleus (npEW) is strongly down-regulated. Therefore, we hypothesized that an altered activity of Ucn1 neurons in the npEW would contribute to the pathogenesis of major depression. To test this hypothesis we measured Ucn1 mRNA and BDNF mRNA levels in the npEW of seven male and four female, drug-free suicide victims with major depression, and compared the data with those obtained from 10 male and seven female individuals without neurological and psychiatric disorders (controls). We show that compared with controls, the Ucn1-mRNA level in npEW neurons is about 9.12 times higher in male but unchanged in female suicide victims. Furthermore, BDNF mRNA expression in microdissections of npEW was 3.36 times lower in male suicide victims, but 5.27 times higher in female victims, compared with controls. Our data also show that male suicide victims had almost 11.47 times more Ucn1 and 4.26 times less BDNF mRNA in the npEW than female suicide victims. We discuss the significance of these data for npEW Ucn1 and BDNF, and propose that altered expressions of Ucn1 and BDNF in the npEW contribute to the pathogenesis of major depression and/or suicidality in a gender-specific manner.

Regional distribution and effects of postmortal delay on endocannabinoid content of the human brain.

Tissue levels of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have been determined in 16 regions and nuclei from human brains, using liquid chromatography/in-line mass spectrometry. Measurements in brain samples stored at -80 degrees C for 2 months to 13 years indicated that endocannabinoids were stable under such conditions. In contrast, the postmortal delay had a strong effect on brain endocannabinoid levels, as documented in brain samples microdissected and frozen 1-6 h postmortem, and in neurosurgical samples 0, 5, 30, 60, 180 and 360 min after their removal from the brain. The tissue levels of AEA increased continuously and in a region-dependent manner from 1 h after death, increasing about sevenfold by 6 h postmortem. In contrast, concentrations of 2-AG, which were 10-100 times higher in human brain regions than those of AEA, rapidly declined: within the first hour, 2-AG levels dropped to 25-35% of the initial ('0 min') value, thereafter they remained relatively stable. As analyzed in samples removed 1-1.5 h postmortem, AEA levels ranged from a high of 96.3 fmol/mg tissue in the nucleus accumbens to a low of 25.0 fmol/mg in the cerebellum. 2-AG levels varied eightfold, from 8.6 pmol/mg in the lateral hypothalamus to 1.1 pmol/mg in the nucleus accumbens. Relative levels of AEA and 2-AG varied from region to region, with the 2-AG:AEA ratio being high in the sensory spinal trigeminal nucleus (140:1), the spinal dorsal horn (136:1) and the lateral hypothalamus (98:1) and low in the nucleus accumbens (16:1) and the striatum (31:1). The results highlight the pitfall of analyzing endocannabinoid content in brain samples of variable postmortal delay, and document differential distribution of the two main endocannabinoids in the human brain.

Decreased stress responsivity of central and peripheral catecholaminergic systems in aged 344/N Fischer rats.

We investigated the effects of stress on central and peripheral sympatho-adrenal and sympatho-neural functions in healthy, intact young (3-4 mo) and aged (24 mo) male Fischer 344/N rats. Extracellular fluid (ECF) levels of the catecholamines norepinephrine (NE), dihydroxyphenylglycol (DHPG), methoxyhydroxyphenylglycol (MHPG), and dihydroxyphenylacetic acid (DOPAC) were obtained by microdialysis in the paraventricular nucleus (PVN) of the hypothalamus at baseline and during immobilization (IMMO). The baseline levels of these substances were similar in both age groups, and their concentrations increased significantly in response to IMMO. The IMMO-induced increases of NE and MHPG, however, were significantly smaller in old than in young rats. Plasma levels of the catecholamines NE, DHPG, MHPG, DOPAC, dihydroxyphenylalanine (DOPA), epinephrine (EPI), dopamine (DA), and HVA were also determined in young and old rats during IMMO. Basal levels of these substances were significantly higher in old than in young rats. The magnitude of the IMMO-induced increases in the majority of these compounds however, was significantly smaller in old than in young rats. We conclude that, at the basal state, aging in the Fischer rat is associated with normal PVN ECF, but high plasma catecholamine levels; at stress state, however, old rats have substantially lesser activation of their central and peripheral catecholaminergic systems than young rats.

Afferent connections of the subparafascicular area in rat.

The subparafascicular nucleus and the subparafascicular area are the major sites of synthesis of the recently discovered neuropeptide, tuberoinfundibular peptide of 39 residues (TIP39). Better knowledge of the neuronal inputs to the subparafascicular area and nucleus will facilitate investigation of the functions of TIP39. Thus, we have injected the retrograde tracer cholera toxin B subunit into the rostral, middle, and caudal parts of the rat subparafascicular nucleus. We report that the afferent projections to the subparafascicular nucleus and area include the medial prefrontal, insular, and ectorhinal cortex, the subiculum, the lateral septum, the anterior amygdaloid area, the medial amygdaloid nucleus, the caudal paralaminar area of the thalamus, the lateral preoptic area, the anterior, ventromedial, and posterior hypothalamic nuclei, the dorsal premamillary nucleus, the zona incerta and Forel's fields, the periaqueductal gray, the deep layers of the superior colliculus, cortical layers of the inferior colliculus, the cuneiform nucleus, the medial paralemniscal nucleus, and the parabrachial nuclei. Most of these regions project to all parts of the subparafascicular nucleus. However, the magnocellular subparafascicular neurons, which occupy the middle part of the subparafascicular nucleus, may not receive projections from the medial prefrontal and insular cortex, the medial amygdaloid nucleus, the lateral preoptic area, and the parabrachial nuclei. In addition, double labeling of cholera toxin B subunit and TIP39 revealed a remarkable similarity between input regions of the subparafascicular area and the brain TIP39 system. Neurons within regions that contain TIP39 cell bodies as well as regions that contain TIP39 fibers project to the subparafascicular area. Overall, the afferent connections of the subparafascicular nucleus and area suggest its involvement in central reproductive, visceral, nociceptive, and auditory regulation.

Forebrain projections of tuberoinfundibular peptide of 39 residues (TIP39)-containing subparafascicular neurons.

Neurons containing tuberoinfundibular peptide of 39 residues (TIP39) constitute a rostro-caudally elongated group of cells in the posterior thalamus. These neurons are located in the rostral part of the subparafascicular nucleus and in the subparafascicular area, caudally. Projections of the caudally located TIP39 neurons have been previously identified by their disappearance following lesions. We have now mapped the projections of the rat rostral subparafascicular neurons using injections of the anterograde tracer biotinylated dextran amine and the retrograde tracer cholera toxin B subunit, and confirmed the projections from more caudal areas previously inferred from lesion studies. Neurons from both the rostral subparafascicular nucleus and the subparafascicular area project to the medial prefrontal, insular, ecto- and perirhinal cortex, nucleus of the diagonal band, septum, central and basomedial amygdaloid nuclei, fundus striati, basal forebrain, midline and intralaminar thalamic nuclei, hypothalamus, subthalamus and the periaqueductal gray. The subparafascicular area projects more densely to the amygdala and the hypothalamus. In contrast, only the rostral part of the subparafascicular nucleus projects significantly to the superficial layers of prefrontal, insular, ectorhinal and somatosensory cortical areas. Double labeling showed that anterogradely labeled fibers from the rostral part of the subparafascicular nucleus contain TIP39 in many forebrain areas, but do not in hypothalamic areas. Injections of the retrograde tracer cholera toxin B subunit into the lateral septum and the fundus striati confirmed that they were indeed target regions of both the rostral subparafascicular nucleus and the subparafascicular area. In contrast, TIP39 neurons did not project to the anterior hypothalamic nucleus. Our data provide an anatomical basis for the potential involvement of rostral subparafascicular neurons in limbic and autonomic regulation, with TIP39 cells being major subparafascicular output neurons projecting to forebrain regions.

The vasopressin 1b receptor is prominent in the hippocampal area CA2 where it is unaffected by restraint stress or adrenalectomy.

The vasopressin 1b receptor (Avpr1b) is one of two principal receptors mediating the behavioral effects of vasopressin (Avp) in the brain. Avpr1b has recently been shown to strongly influence social forms of aggression in mice and hamsters. This receptor appears to play a role in social recognition and motivation as well as in regulating the hypothalamic-pituitary-adrenal axis. Most of these studies have been performed in knockout mice, a species in which the localization of the Avpr1b has not been described, thus precluding correlations with the behaviors. We performed in situ hybridization histochemistry (ISHH) with specific probes and found especially prominent expression within the CA2 pyramidal neurons of the hippocampus, with much lower expression in the hypothalamic paraventricular nucleus and amygdala. Reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed expression in those as well other areas in which the ISHH was not sensitive enough to detect labeled cells (e.g. piriform cortex, septum, caudate-putamen and lower brainstem areas). Mouse Avpr1b transcript levels were not altered in the CA2 field by restraint stress or adrenalectomy. Finally, ISHH and RT-PCR showed expression of the Avpr1b gene in the rat and human hippocampi as well. We suggest that the CA2 field may form or retrieve associations (memories) between olfactory cues and social encounters.

IP3 type 1 receptors in the heart: their predominance in atrial walls with ganglion cells.

Previously we have shown that inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are abundantly expressed in the atria of rat hearts. Since arrangement of atria is very heterogeneous, in this work we focused on the precise localization of IP3 receptors in the left atrium, where the gene expression of the type 1 IP3R was the highest. The mRNA levels of the IP3 type 1 receptors in the left atrium, left ventricle and myocytes were determined using real-time polymerase chain reaction and Taqman probe. For precise localization, immunohistochemistry with the antibody against type 1 IP3Rs was performed. The mRNA of type 1 IP3 receptor was more than three times higher in the left atrium than in the left ventricle, as determined by real-time PCR. Expression of the type 1 IP3 receptor mRNA was higher in the atria, especially in parts containing cardiac ganglion cells. The atrial auricles, which are particularly free of ganglion cells, and the ventricles (wall of the right and left ventricle and ventricular septum) contained four to five times less IP3 receptors than atrial samples with ganglia. IP3R type 1 immunoreactivity detected by a confocal microscope attributed the most condensed signal on ganglionic cells, although light immunoreactivity was also seen in cardiomyocytes. These results show that type 1IP3 receptors predominate in intrinsic neuronal ganglia of cardiac atria.

Anxiolytic 2,3-benzodiazepines, their specific binding to the basal ganglia.

Over the past 20 years, several members of the 2,3-benzodiazepine family have been synthesized. Some of these compounds--tofisopam (Grandaxin), girisopam, nerisopam--exert significant anxiolytic and antipsychotic activities. Sites where actions of 2,3-benzodiazepines are mediated differ from those of 1,4-benzodiazepines. Binding of 2,3-benzodiazepines to neuronal cells in the central nervous system shows a unique and specific distribution pattern: their binding sites are located exclusively to the basal ganglia. Chemical lesioning of the striato-pallido-nigral system, surgical transections of the striato nigral pathway and the activation of c-fos expression in the basal ganglia after application of 2,3-benzodiazepines suggest that these compounds mainly bind to projecting neurons of the striatum. The binding sites are transported from the striatum to the substantia nigra and the entopeduncular nucleus. Recent studies on mechanism of action of 2,3-benzodiazepines indicate their possible role in opioid signal transduction since 2,3-benzodiazepines augment the agonist potency of morphine to induce catalepsy and analgesia, and their action is diminished in morphine tolerant animals. The possible biochemical target of 2,3-benzodiazepines is an alteration in the phosphorylation of protein(s) important in the signal transduction process. Agents affecting emotional responses evoked by endogenous opioids without danger of tolerance and dependence may represent a new therapeutic tool in the treatment of addiction and affective disorders.

Septamer element-binding proteins in neuronal and glial differentiation.

Differentiation of progenitors into neurons and glia is regulated by interactions between regulatory DNA elements of neuron- and glia-specific genes and transcription factors that are differentially expressed by progenitors at progressive stages of neural development. We have identified a novel DNA regulatory element (TTTGCAT = septamer) present on the enkephalin (ENK), neuronal cell adhesion molecule, neurofilament of 68 kDa (NF68), growth-associated protein of 43 kDa, glial high-affinity glutamine transporter, tyrosine hydroxylase, etc., genes. When septamer function was blocked by introducing septamer competitor DNA into primary differentiating neural cultures, mRNA levels of ENK, NF68, and glial fibrillary acidic protein decreased by 50-80%, whereas no effect was seen using a control DNA. Septamer elements serve as binding sites for lineage-specific multimeric complexes assembled from three distinct nuclear proteins. Progenitors express a 16 kDa protein (p-sept) which binds to DNA as a homodimer (detected as the 32 kDa P-band). Cells that entered the neuronal lineage express an additional 29 kDa protein (n-sept) that binds to the homodimerized p-sept, and together they form a 62 kDa multimer (detected as N-band). Cells that entered the glial lineage express a distinct 23 kDa protein (g-sept), which along with the homodimerized p-sept form a 56 kDa multimer (observed as G-band). The binding of the distinct protein complexes (P, G, and N) to the septamer site causes a lineage-specific DNA bending (P = 53 degrees; G = 72 degrees; and N = 90 degrees ), which may contribute to the regulatory effect of the septamer interaction. In summary, septamer and its binding proteins represent novel protein-DNA interactions that may contribute to the regulation of neuroglial differentiation in the developing mammalian CNS.