Gut, Microbiome, and Brain Regulatory Axis: Relevance to Neurodegenerative and Psychiatric Disorders.

It has become apparent that the molecular and biochemical integrity of interactive families, genera, and species of human gut microflora is critically linked to maintaining complex metabolic and behavioral processes mediated by peripheral organ systems and central nervous system neuronal groupings. Relatively recent studies have established intrinsic ratios of enterotypes contained within the human microbiome across demographic subpopulations and have empirically linked significant alterations in the expression of bacterial enterotypes with the initiation and persistence of several major metabolic and psychiatric disorders. Accordingly, the goal of our review is to highlight potential thematic/functional linkages of pathophysiological alterations in gut microbiota and bidirectional gut-brain signaling pathways with special emphasis on the potential roles of gut dysbiosis on the pathophysiology of psychiatric illnesses. We provide critical discussion of putative thematic linkages of Parkinson's disease (PD) data sets to similar pathophysiological events as potential causative factors in the development and persistence of diverse psychiatric illnesses. Finally, we include a concise review of preclinical paradigms that involve immunologically-induced GI deficits and dysbiosis of maternal microflora that are functionally linked to impaired neurodevelopmental processes leading to affective behavioral syndromes in the offspring.

Endogenous morphine: up-to-date review 2011.

Positive evolutionary pressure has apparently preserved the ability to synthesize chemically authentic morphine, albeit in homeopathic concentrations, throughout animal phyla. Despite the establishment of a progressively rigorous and mechanistically focused historical literature extending from the mid 1970s to the mid 1980s that supported the expression of chemically authentic morphine by animal cellular and organ systems, prejudicial scepticism and early dismissal by scientists and clinicians most often obscured widespread acceptance of the biological importance and medical implications of endogenous morphine. The current critical paper presents and evaluates key recent coordinated studies in endogenous morphine research, highlighting those that have advanced our understanding of the functional roles of cognate alkaloid-selective µ(3) and µ(4) opiate receptors. We propose that the expression of endogenous morphine by animal and human cells is designed to mediate homeopathic regulation of metabolic activity via activation of cognate µ(3) and µ(4) receptors that serve as transductive conduits for shortcircuit Ca(++) fluxes. The implications of endogenous morphine coupling to nitric oxide regulation of mitochondrial function, with special reference to the cardiovascular system, are now formulated after many years of neglect.

Psychiatric implications of endogenous morphine: up-to-date review.

For over 30 years empirical studies have repeatedly demonstrated that the biosynthesis of morphine by diverse animal and human tissues occurs. Recently, the blue mussel's neural tissues and human white blood cells were used to demonstrate the de novo biosynthesis of morphine for small precursor molecules derived from the aromatic amino acid L-tyrosine. Because catecholamine precursors, i.e., L-3,4-dihydroxyphenylalanine (L-DOPA), were also found to be utilized as morphine precursors, a novel reciprocally interactive mechanism is apparent that links catecholamine and opioid pathways in the activation and inhibition of diverse tissue responses. Additionally, these observations provide new insights into morphinergic signalling that transcend analgesia and addiction. We have also linked the biological effects of nitric oxide into a common effect in endogenous morphine signalling. Given the singular importance of dopamine and morphine's interaction in the CNS, the presence and association of this signalling with nitric oxide all promises to provide novel answers for mental health phenomena, which have been lacking because of the inability in accepting the empirical endogenous morphine studies.

Cholinergic regulation of morphine release from human white blood cells: evidence for a novel nicotinic receptor via pharmacological and microarray analysis.

Recent work from our laboratory has demonstrated that human white blood cells make morphine and that substances of abuse, i.e. nicotine, alcohol and cocaine have the ability to release this endogenous substance, suggesting a common mechanism of action. We now demonstrate that the nicotinic process is more complex than formerly envisioned. The incorporation rate of 125I-labeled morphine into PMN and MN are 7.85+/-0.36%, 1.42+/-0.19%, respectfully, suggesting in MN this process is of low activity. Separate incubations of PMN with varying concentrations of nicotine or the nicotine agonist epibatidine resulted in a statistically significant enhancement of 125I-trace labeled morphine released into the extracellular medium. In order to ascertain the specificity of the nicotine stimulated morphine release the following experiments were performed. Co-incubation of hexamethonium dichloride (5 microg/ml and at 10 microg/ml), which preferentially blocks nicotinic receptors at autonomic ganglia, with nicotine, exerted a very weak inhibitory effect. Co-incubation of alpha-BuTx or atropine or chlorisondamine diiodide or dihydro-Beta-erythroidine hydrobromide, an alpha4Beta2 receptor antagonist, did not block nicotine induced morphine release alone or in combination, suggesting either the response was not specific or it was mediated by a novel nicotinic receptor. Human leukocyte total RNA isolated from whole blood were analyzed, using the Human Genome Survey microarray (Applied Biosystems), for cholinergic receptor expression. PMN nicotinic receptor gene expression was present and contained numerous variants (eight). The number of variants suggests that indeed a novel nicotinic receptor may be mediating this effect, while simultaneously demonstrating the significance of the cholinergic receptor expression in these immune cells.

Calcium currents in a pituitary cell line (AtT-20): differential roles in stimulus-secretion coupling.

The purpose of the present investigation was to identify voltage-dependent calcium channel subtypes that control the release of ACTH in AtT-20 cells, a clonal mouse pituitary cell line. Using the perforated patch-clamp technique, we identified dihydropyridine (nimodipine)-, omega-Agatoxin IVA-, and omega-Conotoxin MVIIC-sensitive calcium currents. No omega-Conotoxin GVIA-sensitive currents are present in these cells. There also existed a considerable resistant component to the recorded inward current that was inhibited by cadmium, a nonselective calcium channel antagonist. Using RIA, we examined the contributions of each of the pharmacologically distinct calcium channel populations to CRH- or potassium chloride (KCI)-stimulated release of ACTH at various time intervals (10 sec to 60 min). We found that nimodipine markedly inhibited ACTH release at all intervals tested, whereas omega-Agatoxin IVA, omega-Conotoxin MVIIC, and omega-Conotoxin GVIA had no significant effect. Moreover, the inhibition by nimodipine was comparable to that seen after cadmium application, and the effects of these two antagonists were not additive. These data suggest that although AtT-20 cells possess dihydropyridine-, omega-Agatoxin IVA-, and omega-Conotoxin MVIIC-sensitive calcium channels as well as a considerable toxin-resistant current, only the dihydropyridine-sensitive calcium channels appear to be coupled to CRH- or KCI-induced ACTH release.

Substance P and catecholaminergic expression in neurons of the hamster main olfactory bulb.

A coordinated series of immunohistochemical and biochemical analyses have been conducted in the hamster to examine the dependence of substance P and tyrosine hydroxylase (TH) expression by second-order olfactory neurons, and the level of dopamine in the main olfactory bulb (MOB), on the integrity of carnosine- and olfactory marker protein (OMP)-containing primary afferent neurons. Substance P-like immunoreactivity (SPLI) is localized in external tufted cells and centrifugal afferents of the MOB; TH immunoreactivity has a wider distribution, in external tufted, middle tufted, periglomerular, and deep short-axon cells as well as in centrifugal afferents. To characterize the SPLI, this material was isolated by guanidine-HCl extraction and passage over a C18 SEP-PAK. The SPLI coelutes on HPLC with authentic substance P and, following oxidation, coelutes with substance P sulfoxide. It is sensitive to alpha-chymotrypsin and is resistant to trypsin. Thus, the SPLI in the MOB behaves as authentic substance P. Intranasal irrigation with 0.17 M ZnSO4 results in peripheral deafferentiation of the MOB for up to 8 months as evidenced by a persistent loss of OMP immunoreactivity and shrinkage of the olfactory nerve layer and glomeruli. By these criteria, the vomeronasal inputs to the accessory olfactory bulb are not destroyed and the spared vomeronasal receptor neurons do not innervate the vacated peripheral projection field in the MOB. The loss of peripheral inputs to the MOB is accompanied by marked and parallel reductions in the incidences of SPLI- and TH-positive second-order neurons despite an increase in the density of neuronal somata in the glomerular layer. Biochemical quantifications following peripheral deafferentation also demonstrate significant decreases of both substance P and dopamine, together with the expected decrease of carnosine. In contrast, the SPLI and the TH and serotoninlike immunoreactivities in centrifugal afferents as well as the TH immunoreactivity in deep interneurons do not appear to be reduced, and the MOB content of norepinephrine in centrifugal afferents is unaffected. These results collectively indicate that the loss of inputs from the primary olfactory receptor neurons can reduce the levels of at least two different, putatively neuroactive compounds (substance P and dopamine) in at least three classes of second-order neurons (external tufted, middle tufted, and periglomerular cells). The control of central neuron phenotype by the peripheral olfactory neurons thus appears to be a phenomenon of broad influence. It may play a role in processing chemosensory information as well as offering a system in which to study neuronal plasticit

Alterations in neuropeptide Y, tyrosine hydroxylase, and Y-receptor subtype distribution following spinal nerve injury to rats.

Recent animal models of experimental nerve injury have proven useful in evaluating potential sympathetic involvement in neuropathic pain syndromes. We have employed a widely adopted unilateral L5/L6 spinal nerve ligation model to compare the development of mechanical allodynia with neurochemical changes both at the site of peripheral nerve injury and in the dorsal root ganglia (DRG). We have focused on the expression of neuropeptide Y (NPY), a well-studied regulatory peptide and phenotypic marker of sympathetic neurons, and functionally related Y-receptor binding sites following nerve injury. In sympathetic neurons, NPY is colocalized and coreleased with norepinephrine (NE) at peripheral sites of action. Furthermore, NPY gene expression is induced within the population of medium- and large-diameter DRG neurons of the A beta-fiber class after experimental nerve injury. We therefore hypothesized that concurrent alterations in NPY and NE expression by sympathetic and sensory neurons may be a contributing factor to sympathetically-maintained neuropathic conditions. Animals with unilateral L5/L6 spinal nerve ligation developed mechanical allodynia of the hind paw ipsilateral to the site of injury that persisted until sacrifice at postoperative day 10. A significant induction of preproneuropeptide Y-encoding (PPNPY) mRNA, as detected by in situ hybridization histochemistry (ISHH), occurred in populations of medium- and large-diameter DRG neurons ipsilateral to the site of injury. Immunohistochemical analysis indicated a marked decline in the number of labeled sympathetic axons positive for tyrosine hydroxylase-like and NPY-like immunoreactivities (TH-LI and NPY-LI, respectively) proximal to the site of nerve injury and almost complete elimination of immunopositive fibers distal to the site of ligation. Whereas, the extent of colocalization of NPY-LI to TH-LI-positive sympathetic axons in unaffected L4 or L5 nerve segments exceeded 80%, this figure declined to approximately 50% in regenerating axons of ligated spinal nerve L5. The portion of NPY-LI that was not colocalized to sympathetic TH-LI-positive fibers was most likely contributed by regenerating sensory axons, consistent with marked de novo synthesis of NPY by DRG neurons. In end bulb axon terminals, i.e. morphological profiles characteristic of neuromas, NPY-LI-positive elements that were not colocalized to TH-LI-positive sympathetic elements appeared to be spatially segregated from those of sympathetic origin with colocalized TH-LI and NPY-LI. Receptor autoradiography indicated that small- and medium-diameter DRG somata of the C-fiber class normally express both Y1 and Y2 receptor subtypes. The pattern of the distribution of Y-receptor binding sites appeared to be relatively unaffected by spinal nerve ligation. In contrast, there was a marked increase in the density of Y2 receptor binding sites in the proximal segment of ligated spinal nerve L5, consistent with previously published data indicating differential transport of the Y2 autoregulatory receptor subtype to nerve terminals. Induction of NPY gene expression in injured DRG neurons is consistent with appearance of NPY-LI-positive end bulbs derived from regenerating sensory axons that are found in developing neuromas containing a relatively high density of transported prejunctional Y2 receptors. Newly established functional interactions of spatially segregated sensory- and sympathetically-derived end bulbs in developing neuromas may enhance neuronal hyperexcitability engendered by aberrant electrical activity at the site of injury. Injury-related alterations in the regulatory activities of NPY released within the DRG at somally-distributed Y-receptors may also contribute to the development and/or persistence of symptoms characteristic of sympathetically-maintained pain. Finally, at later times NPY-mediated modulation of NE release from invading sympathetic axon terminals within the DRG may affect the extent of alpha2 rece

Selective in situ hybridization histochemical analyses of alternatively spliced mRNAs encoding beta- and gamma-preprotachykinins in rat central nervous system.

The present study describes the development of an in situ hybridization histochemistry (ISHH) procedure which was employed to selectively monitor cellular distributions of the 2 major alternatively spliced beta- and gamma-species of mRNA encoding preprotachykinin (PPT) molecules found in rat CNS. For these purposes, 2 custom-designed oligodeoxynucleotide probes were synthesized corresponding to complementary sequences of beta- and gamma-PPT mRNAs. In particular, the gamma-selective probe was demonstrated to hybridize to the contiguous regions of RNA flanking the splice site formed by exclusion of exon 4. Initially, Northern blot analyses performed in conjunction with appropriate specificity controls demonstrated selective hybridization of the 32P-labeled beta- and gamma-selective probes to single bands of approximately 1.2-1.3 kilobases in size, consistent with previously established values for rat brain beta- and gamma-PPT mRNAs. In anatomical studies, results obtained from absorptions using competing nonradiolabeled oligonucleotides defined the specificity and selectivity of both probes for targeting their respective species of mRNA immobilized within sections of brain tissue. Extensive ISHH analyses using both beta- and gamma-selective probes demonstrated similar patterns of cellular labeling in all of the examined CNS areas. In addition, data obtained from analyses of adjacent thin sections of the dorsal root ganglia (DRG) indicated that beta- and gamma-PPT mRNAs were colocalized within individual DRG neurons, thereby suggesting generalized coexpression at the cellular level of both forms of mRNA. These data were complemented by semi-quantitative analyses which yielded cellular or intrinsic molar ratios of beta- to gamma-PPT mRNA of approximately 1:2-1:3, consistent with those values previously determined by nuclease protection analyses. In sum, a reasonable hypothesis evolving from the anatomical studies in combination with previous biochemical data supports the existence of a strong homeostatic mechanism involved in the maintenance of relatively constant intrinsic molar ratios of beta- to gamma-PPT mRNA by tachykinin-expressing neurons. The biological relevance of this putative fundamental relationship is discussed in the context of posttranslational processing of PPT molecules and of expression of mature tachykinins.

Behavioral sensitization to cocaine after a brief social defeat stress: c-fos expression in the PAG.

The experiments explored the nature and time course of changes in behavior and Fos expression in the periaqueductal grey area (PAG) in response to an injection of cocaine that was given following a single episode of social defeat stress. Social defeat stress was defined as an intruder mouse's response to an aggressive resident mouse. First, the intruder was briefly attacked, and secondly, it was threatened while protected by a perforated cage for 20 min. Plasma corticosterone levels rose after the beginning of the confrontation and remained elevated during the protected phase. In a first experiment, separate groups of intruder and control mice were challenged once with cocaine (20, 30, or 40 mg/kg) or saline. During tests for motor activity, behavioral measurements were obtained via (1) photobeam interruptions, (2) tracking of movements via image analysis, and (3) quantitative ethological analysis of postures and acts via videorecords. Several indices of ambulatory or horizontal forward locomotion confirmed the stimulant effects of cocaine. In a further experiment, separate groups of mice were challenged with 40 mg/kg cocaine at one time point, either during the social stress or 3, 5, 7 or 9 days thereafter. A cocaine challenge significantly increased locomotion 5 and 7 days after a brief social defeat stress, in excess of the level that is seen in non-stressed animals. Further experiments used immunohistochemical assays of sections through the caudal ventrolateral PAG and showed a significant increase in Fos-like immunoreactivity (Fos-LI) 1 h after the social stress experience or after cocaine. Importantly, concurrent administration of cocaine with social defeat stress produced inhibition of Fos expression throughout the PAG. A partial to complete recovery of cocaine-induced Fos expression was observed 5-7 days after social defeat stress. The results suggest that a single social stress episode is sufficient to engender a delayed sensitization of stimulant hyperactivity. The initial inhibition of Fos expression by concurrent social stress and cocaine may point to a relevant initiating event in the process of sensitization to stimulants.

Substance P immunoreactivity in the superficial laminae of the hamster olfactory bulb.

The direct application of colchicine to the hamster olfactory bulb has engendered the appearance of a broader distribution of substance P-like immunoreactivity (SP-LI) than has been previously reported. In the glomerular layer, both external tufted and periglomerular cells were strongly immunopositive. These two classes could be identified by somal size and the presence and branching pattern of intraglomerular dendrites. SP-LI neurons were also identified in the external plexiform layer and many were similar to middle tufted cells. These findings demonstrate that substance P expression is not uniquely associated with classes of projection neurons, but is also found in periglomerular cells, a class of inhibitory local circuit neurons.

Social defeat stress increases expression of mu-opioid receptor mRNA in rat ventral tegmental area.

Prompted by previous studies linking social defeat stress to changes in opioid antinociception, we evaluated the expression of mu-opioid receptor (MOR)-encoding mRNA in selected rat brain areas as a function of this type of stress. Because opioids mediate significant regulatory activities of brain dopamine neurons, dopaminergic loci in the ventral tegmental area (VTA) and substantia nigra (SN) were selected for analysis. Within 30 min after social defeat stress, the level of MOR-encoding mRNA, as detected and quantified by in situ hybridization histochemistry, increased in the lateral VTA and this increase was present for at least 6 h. In contrast, defeat stress was without effect on the expression of MOR-encoding mRNA in the SN. These data suggest that stress-induced alteration of MOR-encoding mRNA expression in the VTA may be involved in the consequences of social defeat stress.

Reduced levels of substance P in the brains of Cpe(fat)/Cpe(fat) mice.

This study examines the role of carboxypeptidase E (CPE) in processing pro tachykinin to form the final bioactive amidated undecapeptide, substance P (SP) in various rat brain regions. Cpe(fat)/Cpe(fat) mice brain tissue was analyzed for total SP forms (including intermediates), and final amidated SP was compared to Cpe+/Cpe+ and Cpe+/Cpe- controls. In all brain regions tested by radioimmunoassay, amidated fully processed SP was more than fivefold lower in Cpe(fat)/Cpe(fat) mice than in controls whereas total SP species levels were unchanged. This demonstrates that CPE is required for normal SP proteolytic processing. Substance P has numerous functions in the brain; therefore, SP deficiency due to the CPE mutation may contribute to the obese phenotype or even to other phenotypes not yet described in Cpe(fat)/Cpe(fat) mice.

Streptozotocin-induced diabetes is associated with altered expression of peptide-encoding mRNAs in rat sensory neurons.

Major complications arising from diabetes mellitus include neuropathic pain and altered peripheral inflammatory responses. Somatostatin (SOM), calcitenin gene-related peptide (CGRP), and substance P (SP) are neuropeptides that modulate pain responses transmitted by primary sensory afferents, the cell bodies of which are located in the dorsal root ganglion (DRG). Thus, the goal of the present study was to determine whether the diabetic condition is associated with altered neuropeptide gene expression in lumbar DRG of the rat. We employed an established animal model in which streptozotocin (STZ, 55 mg/kg) is administered to 6 week-old rats. The hallmark symptoms of hyperglycemia (blood glucose > 400 mg/dl), polydipsia, polyuria, and severe weight loss were maximal at 6 weeks postadministration, at which time animals were sacrificed. For determination of peptide encoding mRNAs distributed in DRG neurons, in situ hybridization histochemistry utilizing S-end-labeled oligonucleotides complimentary to sequences of preprosomatostatin (PPSOM), preprocalcitonin gene related peptide (PPCGRP), preprotachykinin (PPT), or preproneuropeptide Y (PPNPY) mRNA was performed. Silver grains were detected overlying DRG cells by autoradiography on sections of tissue counterstained with thionin. Semiquantitative analysis of differences in silver grain signal were made using an image analysis system, which expressed signals as fCi/microns2. In diabetic rats there was a significant decrease in DRG PPSOM (54%, p < 0.01), and PPCGRP (33%. p < 0.05) mRNA hybridization from the normal values PPT mRNA hybridization signal and SP-like immunoreactivity were not significantly changed in diabetic rat DRGs compared to control. In contrast, there was an increase in the number of cells labeled with PPNPY hybridization in DRG from diabetic rats. These data suggest that CGRP and SOM synthesis in primary sensory neurons is reduced in STZ-induced diabetic rats. These changes could contribute to the painful neuropathies and altered inflammatory responses seen in diabetes mellitus.

Immediate-early gene expression in ovine brain after hypothermic circulatory arrest: effects of aptiganel.

BACKGROUND: Altered gene expression occurs in the brain after global ischemia. We have developed a model to examine the effects of cardiopulmonary bypass and hypothermic circulatory arrest (HCA) on the induction of the immediate-early gene c-fos in the brains of neonatal lambs. We then tested the effects of the noncompetitive N-methyl-D-aspartate antagonist, aptiganel hydrochloride (Cerestat), on c-fos expression and neuronal injury. METHODS: Neonatal lambs (weight, 4 to 6 kg) anesthetized with isoflurane were supported by cardiopulmonary bypass, subjected to 90 or 120 minutes of HCA at 15 degrees C, and rewarmed on bypass to 38 degrees C. One hour after cardiopulmonary bypass was terminated, the brains were perfusion fixed and removed for in situ hybridization and immunohistochemical analysis. Some animals survived 3 days before their brains were removed to examine for neuronal necrosis. One group of lambs (n = 20) received aptiganel (2.5 mg/kg). A second group (n = 25) received saline vehicle only. RESULTS: Increasing duration of HCA induced a corresponding increase in c-fos messenger RNA expression throughout the hippocampal formation and cortex. However, Fos protein synthesis peaked after 90 minutes of HCA and decreased significantly (p < 0.01) after 120 minutes of HCA. Aptiganel administration caused a significant decrease in (p < 0.001) c-fos messenger RNA expression and Fos protein synthesis after 90 minutes of HCA and preserved Fos protein synthesis after 120 minutes of HCA. Neuronal necrosis was observed in the brains of vehicle-treated lambs after 120 minutes of HCA but was significantly decreased (p < 0.05) in the lambs given aptiganel. CONCLUSIONS: These experiments indicate that the transcriptional processes of immediate-early genes remain intact, whereas translational processes are impaired after prolonged HCA. The inability to synthesize Fos proteins after 120 minutes of HCA was associated with neuronal degeneration. Aptiganel preserved translational processes and caused a significant improvement in the neurologic outcome.

Distribution of tachykinin- and opioid-expressing neurons in the hamster solitary nucleus: an immuno- and in situ hybridization histochemical study.

In several sensory systems, tachykinin- and opioid-expressing neurons functionally interact and influence the processing of afferent information. To determine whether a similar relationship exists for the processing of general and special (gustatory) visceral afferent information, the present study mapped the distributions of these two neuronal phenotypes within the nucleus of the solitary tract (NST) of the hamster by employing a combination of immuno- and in situ hybridization histochemistry (ISHH). The hamster was chosen because it is frequently used as a model in taste studies, yet there is a relative dearth of data about peptide expression or the classical neurotransmitters in the brainstem of this animal. The immunohistochemical analyses employed 2 highly selective antisera directed towards the prototypical tachykinin and opioid peptides, i.e. substance P (SP) and methionine enkephalin (ENK), respectively. Intense staining of fibers and preterminal/terminal puncta was concentrated in the rostral pole or gustatory zone of the NST. SP-, but not ENK-like immunoreactivity was also observed in long courses of axon bundles traversing the brainstem enroute to the NST. Local application of colchicine engendered the appearance of a moderate number of SP-positive somata that were mostly clustered in the medial, central and intermediate subnuclei, as well as being scattered throughout the remainder of the NST, including the gustatory zone. A low number of isolated ENK-positive somata were also observed throughout the NST. The somal areas of the SP- and ENK-positive somata averaged 86.3 and 81.8 microns 2, respectively. The ISHH studies were performed using 2 selective oligodeoxynucleotide probes with complementary sequences to mRNAs encoding gamma-preprotachykinin (PPT) and preproenkephalin (PPE) molecules. Overall, the cellular expression of PPT mRNA within the NST corresponded both in distribution and in number to those identified by immunohistochemical analyses using anti-SP serum. In contrast, ISHH analyses monitored a significantly greater number of PPE-expressing somata in the medial, central, intermediate and ventrolateral nuclei than were ENK immunoreactive. These findings indicate that tachykinin and opioid peptide phenotypes are represented in neurons throughout the hamster NST and suggest a functional role for PPT- and PPE-related peptide forms in the modulation of afferent general visceral and gustatory information.

Biochemical characterization and anatomical distribution of a major form of unamidated precursor of substance P in rat brain.

Previous work from this laboratory has provided biochemical characterization of several posttranslational processing intermediates of the neuropeptide substance P (SP) in central nervous system (CNS) tissues, including the COOH-terminal glycine-extended dodecapeptide Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly (SP-G). SP-G is a major species of unprocessed SP found in rodent CNS tissues, and is the likely immediate precursor form of SP in the biosynthetic scheme. Here we present extensive characterization of the normal regional distribution of SP-G, as compared to SP, throughout the rat CNS via coordinated biochemical and morphological analyses. By radioimmunoassay (RIA), an approximate 10-fold variation in regional levels of SP-G-like immunoreactivity (SP-G-LI) was observed, ranging from 0.30 pmol/g in the amygdala, to 6.49 pmol/g in the medulla. On a normalized basis, the regional variation of unamidated precursor relative to mature peptide (SP-G-LI/SP-LI molar ratio) ranged from 0.30% in the amygdala to 5.15% in the dorsal root ganglia (DRG). Overall, the highest SP-G-LI/SP-LI ratios were found in DRG, medulla, and spinal cord, i.e. CNS areas associated with primary sensory afferent innervation via capsaicin-sensitive unmyelinated small diameter fibers. In addition, chromatographic and RIA analyses of extracted brain tissues indicated that the quantified immunoreactivities corresponding to SP, SP-G, as well as an additional COOH-terminal Gly-Lys-extended precursor, i.e., SP-G-K, displayed very similar chromatographic behavior as demonstrated for chemically authentic standards. These biochemical data were complemented by immunohistochemical analyses demonstrating a pattern of immunohistochemical staining for the presence of SP-G-LI as a defined subset of SP-LI-containing neural elements. Here, reaction product was localized to dendritic, axonal, and terminal neuronal elements in representative CNS regions of the rat, with relatively high levels of SP-G-LI found within anatomical areas containing a high density of sensory terminal structures. In an attempt to provide correlative functional anatomy, a group of rats was treated with colchicine, in order to differentially localize SP-LI- and SP-G-LI-containing somata after inhibition of axoplasmic transport. Most prominently, colchicine administration engendered immunohistochemical visualization of both SP-LI- and SP-G-LI-positive cells in mesencephalic and brainstem regions associated with stress, pain responses, and central control of autonomic function. Within this context, the coordinate expression of both SP-LI- and of SP-G-LI-positive somata in discrete brain areas is probably indicative of high ongoing rates of tachykinin synthesis coupled to utilization.

Effect of early exposure to delta-9-tetrahydrocannabinol on the levels of opioid peptides, gonadotropin-releasing hormone and substance P in the adult male rat brain.

The effects of neonatal exposure to delta-9-tetrahydrocannabinol (THC) on the adult animal brain neurochemistry and pain perception were evaluated. Newborn rat pups were culled to a litter size of 8 (males and females) and treated either with THC (2 mg/kg) or oil (control) daily, during days 1-4 after birth. After weaning, the THC-treated males were housed 4 per cage. During the juvenile period (day 50), the THC-treated animals exhibited significantly lower baseline tail-flick values (a measure of pain perception) than the control. However, as adults, the THC-treated animals exhibited significantly higher sensitivity to pain following 5 mg/kg morphine challenge. Furthermore, the THC-treated animals had significantly elevated beta-endorphin and methionine-enkephalin levels in almost all the brain areas sampled for the study. In addition, the neonatally THC-treated rats exhibited significantly higher levels of substance P (SP) and significantly lower levels of gonadotropin releasing hormone (GnRH) in the anterior hypothalamus-preoptic area. The SP and GnRH levels did not differ among the THC-treated and control animals in the medial basal hypothalamus. The results of this study indicate that even a very low dose of THC administered during the neonatal period has a long-lasting effect on the brain neurochemistry. In particular, neonatal administration of THC appears to alter functioning of the endogenous opioid system.

Behavioral sensitization to cocaine after a brief social stress is accompanied by changes in fos expression in the murine brainstem.

The objective of the present study was to determine how c-fos gene expression in brainstem structures after a brief episode of social defeat stress is related to behavioral sensitization to cocaine challenge. Social stress was defined as defeat in a brief confrontation with an aggressive resident mouse and subsequent 20-min exposure to the resident's threats behind a protective screen. Mice were treated with cocaine (40 mg/kg, i.p.) immediately or 1 week after social defeat stress. Fos-like immunoreactive (Fos-LI) cell nuclei were analyzed in the ventral tegmental area (VTA), dorsal raphe nucleus (DR), periaqueductal grey area (PAG) and locus coeruleus (LC). One episode of social stress induced behavioral sensitization to cocaine as indicated by an augmented locomotor response to a challenge injection 7 days after a single defeat. In naive mice, social stress markedly increased the number of Fos-LI nuclei in the DR, PAG and LC, but not in the VTA. Similarly, cocaine administration resulted in a significantly increased number of Fos-LI nuclei in the same areas. Administration of cocaine immediately following social defeat significantly reduced the number of Fos-LI nuclei in the DR, PAG and LC. Cocaine-induced Fos expression returned in the PAG and DR, but not in the LC, 1 week after social stress. In conclusion, the present results suggest that the presence of brainstem Fos be related to the ability to express stress-induced behavioral sensitization to cocaine.

The mucosa mediates tachyphylaxis to leukotrienes C4, D4 and E4 in guinea pig trachea.

Effects of mucosal abrasion on tracheal smooth muscle leukotriene C4, D4 and E4 tachyphylaxis and on histamine contractions before and after four leukotriene exposures were studied. In intact tracheae, leukotriene C4 and E4 second exposure contractions were greater than first. Subsequent contractions showed tachyphylaxis. Leukotriene D4 contractions exhibited progressive tachyphylaxis. Mucosal abrasion potentiated initial leukotriene C4 and D contractions and eliminated leukotriene C4, D4 and E4 tachyphylaxis. Four leukotriene (10(-8) M) exposures reduced histamine (10(-4) M) responses in intact but not abraded preparations. Thus leukotriene C4, D4 and E4 release non-specific inhibitor(s) from tracheal mucosa.

Spinal dynorphin involvement in the analgesia of pregnancy: effects of intrathecal dynorphin antisera.

In both rats and humans there is an analgesia associated with pregnancy. This analgesia is spinally mediated and involves the kappa type of opiate receptor. The current study demonstrates that intrathecal administration of high affinity dynorphin antibodies produces a significant reduction in jump thresholds during pregnancy (day 20). The administration of pre-adsorbed antisera fails to produce this effect. These results support the hypothesis that a spinal dynorphin/kappa opiate receptor system is activated during gestation.