Ionotropic Excitatory Amino Acid Research Articles

Kynurenic Acid Levels and Kynurenine Aminotransferase I, II and III Activities in Ganglia, Heart and Liver of Snail Helix Pomatia.

Kynurenic acid (KYNA), a tryptophan metabolite along the kynurenine pathway, is an endogenous antagonist of glutamate ionotropic excitatory amino acid (EAA) receptors and the α7 nicotinic acetylcholine receptor (nAChR). The involvement of KYNA in various pathological conditions and during the aging process is significant. KYNA synthesis from L-kynurenine (L-KYN), through the action of several kynurenine aminotransferases (KATs), is present in the central nervous system (CNS) and periphery of mammals. We were interested in investigating the ability of the brain and peripheral organs of Helix pomatia snails to synthesize KYNA, in an in vitro study. In comparative studies between rat and snail, we looked for the synthesis of KYNA in the liver. We then looked for an effect of age on KYNA synthesis. Ten shell parameters of the Helix pomatia snail were used to establish an Age Rating Scale (ARS), i.e. body weight, shell weight, shell length, width and height, shell opening length and width, lip width, number of shell turns and external shell growth rings. An age of the snails was determined according to the ARS and the snails were divided into three groups, i.e. young, middle and old age. Homogenates of dissected regions, i.e. cerebral ganglia (CG), subpharyngeal ganglia (SG) consisting of pedal, visceral and pleural ganglia, heart and liver, were examined. KYNA was measured by high performance liquid chromatography (HPLC) and KAT activities were measured by an enzymatic method. With respect to ARS, an evaluation of the age of the snails between young (1-2 years), middle (5-7 years) and old (9-13 years) showed significant differences (p<0.001). Analysis of KYNA levels in different snail tissues, i.e. CG, SG, heart and liver, showed an occurrence in the low femtomolar range. Marked and significant increases of KYNA were found in the liver of middle and old age groups. In the SG, KYNA decreased significantly with age. There were no differences in KYNA levels between groups in CG and heart. The lowest KAT activity was found in CG and SG (5 pmol/mg/h), while in heart and liver the values were visibly higher (between 8 and 80 pmol/mg/h). Only in the liver, and exceptionally only for KAT I, the activity increased significantly with age, i.e. up to 14 years. No age-related changes in KAT I, II and III activities were found in CG and SG. Snail liver shows a different pattern of KAT activities compared to the rat liver. Regions of the CNS and periphery of the snail Helix pomatia are able to synthesize KYNA due to KAT activities. In the snail liver, KAT I activity increased with age. Notably, there was no age-related increase in KAT activities in the heart and especially in the CNS of Helix pomatia, indicating significant differences from mammals. A moderate KYNA metabolism in the Helix pomatia snail in the periods studied, up to 14 years, could be a physiological phenomenon that protects organs from possible functional insufficiency due to high KYNA levels, as has been suggested. It is reasonable to search for the factor(s) that could regulate the concentration of KYNA in the body of the snail.

Sympathoexcitation and pressor responses induced by acetate, an ethanol metabolite in the hypothalamic paraventricular nucleus involves activation of NMDA receptors in rats

Alcohol abuse plays a crucial role in the prevalence of cardiovascular and neuronal degenerated diseases. However, the central mechanism that mediates these effects are not fully understood. It has been well know that autonomic hypothalamic paraventricular nucleus (PVN) play an important role in regulating sympathetic outflow and arterial blood pressure (ABP). Moreover, it has been demonstrated that the increased glutamatergic activity among PVN neurons contributed to the sympathoexcitation and development of hypertension. Furthermore, we have reported that ethanol and acetate increase sympathetic outflow and arterial pressure, which may involve the activation of NMDA receptors in autonomic central nucleus of amygdala. Combing with the fact that autonomic PVN neurons are abundantly expressed NMDA receptors, we hypothesize that acetate, the metabolic products from alcohol, activates the PVN neurons through the increase in glutamatergic activity and contributes to the sympathoexcitation and development of chronic diseases, such as hypertension. In anesthetized rats, the effect of acetate microinjected in the PVN on the renal sympathetic nerve activity (RSNA) and arterial blood pressure (ABP) was determined. The PVN acetate microinjection increased sympathoexcitatiory and pressor response in a dose dependent dose-dependent (1.5mm, 0.5mm, 2.0mm, 7.5mm) manner, and 2mm of acetate showed a minimum dose evoked a maximal response. To determine the role of acetate-stimulated glutamatergic receptor activation in driving sympathoexcitatory and pressor responses, either 2mm acetate or pre-treatment of Kynurenic acid (KYN, 7.2mm) ionotropic excitatory amino acid receptor blocker, or D-2-amino-5-phosphopentanoate (AP5, 3.0mm), a N-methyl-D-aspartate (NMDA) receptor antagonist, followed by 2mm acetate were microinjected into the PVN of male Sprague Dawley rats (300-500g, n= 6-9/ group). RSNA and ABP responses were compared among vehicle (saline) and 2mm acetate, pre-treatment of KYN and 2mm acetate, or pre-treatment AP5 and 2mm acetate protocols. 2mm acetate significantly increased RSNA (60-70% baseline, p &lt; 0.001) and mean arterial pressure (MAP, 10-12mmHg, p &lt; 0.05). Non-selective glutamatergic receptor antagonist, KYN significantly blocked the sympathoexcitatory (RSNA, p &lt; 0.05) and pressor response (MAP, p &lt; 0.05) evoked by 2mm acetate in the PVN. Furthermore, selective NMDA receptor antagonist, AP5 significantly attenuates the sympathoexcitatory response induced by PVN 2mm acetate (RSNA, P &lt; 0.05 and MAP, p &lt; 0.01). These data indicate acetate can increase glutamatergic activity and excitability of pre-sympathetic PVN neurons via activation of local NMDA receptors. The metabolism of ethanol to acetate following by alcohol consumption may contribute, in part, to the development of neurogenic hypertension and/or other cardiovascular diseases associated with increased sympathetic outflow. R15HL145655, R15HL150703 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

An origin of carotid vasodilation extends along the full extent of the parasympathetic parvicellular reticular region in the rat brainstem.

During grooming in rats, cranial parasympathetic activation leads to increased carotid artery blood flow, but the brainstem origin of this vasodilation signal is poorly understood. The aim of this study was to map brainstem sites wherein chemical stimulation with l-cysteine, an ionotropic excitatory amino acid receptor activator, can trigger carotid vasodilation in anesthetized intact and superior cervical sympathectomized (SCD) rats. The right side of the brainstem was accessed ventrally; arterial blood pressure and right carotid artery flow resistance were monitored. Employing microinjections of l-cysteine into the ipsilateral brainstem, vasodilation triggering sites were identified dorsal and rostral to the rostral ventrolateral medulla (RVLM) presympathetic pressor area, forming a parasagittal plate-like response zone that varied from rat to rat. In SCD rats (N=6), we observed a vasodilation zone in a region that included the RVLM pressor area, suggesting a parasympathetic origin. A similar zone was identified in intact rats (N=8), except that it did not include part of the RVLM pressor area. Dye injected into vasodilator sites labelled the rostral part of the parvicellular reticular formation (PcRt), consistent with a cranial parasympathetic origin. Employing mapping with L-cysteine, a putative neuromodulator, this study provides the first functional demonstration of a carotid vasodilation area that extends along the full extent of the parasympathetic rostral PcRt region in rats.

Topographic carotid vasoconstriction in the rostral ventrolateral medulla of rats.

The vascular beds of various cranial tissues receive common carotid flow, which contributes to blood flow redistribution associated with animal behaviors such as grooming, but the medullary autonomic regulation of carotid flow resistance (CAR) is poorly understood. This study is the first to examine the response sites of CAR in the rat rostral ventrolateral medulla (RVLM) presympathetic area to chemical stimulation by the ionotropic excitatory amino acid receptors activator L-cysteine. Arterial blood pressure and CAR were monitored in anesthetized rats which had a cranial window constructed above the ventral medulla. Mapping of L-cysteine microinjection in eight rats showed carotid vasoconstriction in the caudal part alone within the RVLM pressor area, which included contributions from other vascular beds, indicating localized topographic carotid vasoconstriction. Additional testing was performed on four types of denervated rats. A similar response map was obtained in six rats that received minimal lesions during surgery as well as in 10 rats with severed internal or external carotid nerves. However, the remaining three minimally lesioned rats showed extensive vasoconstriction of the RVLM pressor area including the rostral part, indicating lack of a topographic response. The topographic response of most rats might be state-dependent. Seven rats with complete cervical denervation showed no carotid vasoconstrictor response in the RVLM pressor area, indicating cervical sympathetic mediation of the responses. The topographic carotid vasoconstriction in response to L-cysteine may suggest differential roles of presympathetic neurons in the rostral and caudal parts of the RVLM in sympathetic carotid flow regulation.

Central cardiovascular actions of L-homocysteine microinjected into ventrolateral medullary autonomic areas of the rat.

Elevated L-homocysteine concentrations in the plasma and cerebrospinal fluid are related to cardiovascular and neuronal diseases, and could contribute to disease development. However, the central cardiovascular actions of L-homocysteine in two important autonomic regulating areas remain unknown: the rostral ventrolateral medulla (RVLM), including pre-sympathetic neurons, and the caudal ventrolateral medulla (CVLM), including interneurons projecting to pre-sympathetic neurons in the RVLM. Therefore, the aim of the current study was to examine the influence of L-homocysteine microinjected into the RVLM and CVLM areas on changes in arterial blood pressure (ABP) and heart rate (HR) of anesthetized rats, as well as the influence of ionotropic excitatory amino acid (iEAA) receptors on the central actions of L-homocysteine. L-Homocysteine solutions were microinjected into the RVLM and CVLM, which were defined according to pressor and depressor responses to L-glutamate microinjections, respectively. ABP and HR increased in the RVLM and decreased in the CVLM after microinjection with L-homocysteine, similar to L-glutamate, in a dose-dependent manner, suggesting mediation of EAA receptors. Prior microinjection of the N-methyl-D-aspartate (NMDA) iEAA receptor antagonist MK801, but not the non-NMDA receptor antagonist CNQX, abolished the observed responses to L-homocysteine in both the RVLM and CVLM. These results indicate the central cardiovascular actions of L-homocysteine via MK801-sensitive receptors of the medullary autonomic neurons in the rat RVLM and CVLM. It remains unknown if the central cardiovascular actions are related to cardiovascular diseases after endogenously and locally augmented L-homocysteine production by disordered metabolism. Further studies on functional significance of L-homocysteine may provide some clue to understand its toxic mechanism.

Sympathoexcitation and pressor responses induced by ethanol in the central nucleus of amygdala involves activation of NMDA receptors in rats.

The central nervous system plays an important role in regulating sympathetic outflow and arterial pressure in response to ethanol exposure. However, the underlying neural mechanisms have not been fully understood. In the present study, we tested the hypothesis that injection of ethanol in the central nucleus of the amygdala (CeA) increases sympathetic outflow, which may require the activation of local ionotropic excitatory amino acid receptors. In anesthetized rats, CeA injection of ethanol (0, 0.17, and 1.7 μmol) increased splanchnic sympathetic nerve activity (SSNA), lumbar sympathetic nerve activity (LSNA), and mean arterial pressure (MAP) in a dose-dependent manner. A cocktail containing ethanol (1.7 μmol) and kynurenate (KYN), an ionotropic excitatory amino acid receptor blocker, showed significantly blunted sympathoexcitatory and pressor responses compared with those elicited by CeA-injected ethanol alone (P < 0.01). A cocktail containing ethanol and d-2-amino-5-phosphonovalerate, an N-methyl-d-aspartate (NMDA) receptor antagonist, elicited attenuated sympathoexcitatory and pressor responses that were significantly less than ethanol alone (P < 0.01). In addition, CeA injection of acetate (0.20 μmol, n = 7), an ethanol metabolite, consistently elicited sympathoexcitatory and pressor responses, which were effectively blocked by d-2-amino-5-phosphonovalerate (n = 9, P < 0.05). Inhibition of neuronal activity of the rostral ventrolateral medulla (RVLM) with KYN significantly (P < 0.01) attenuated sympathoexcitatory responses elicited by CeA-injected ethanol. Double labeling of immune fluorescence showed NMDA NR1 receptor expression in CeA neurons projecting to the RVLM. We conclude that ethanol and acetate increase sympathetic outflow and arterial pressure, which may involve the activation of NMDA receptors in CeA neurons projecting to the RVLM.

Functional cardiovascular action of l-cysteine microinjected into pressor sites of the rostral ventrolateral medulla of the rat

The endogenous sulfur-containing amino acid L-cysteine injected into the cerebrospinal fluid space of the cisterna magna increases arterial blood pressure (ABP) and heart rate (HR) in the freely moving rat. The present study examined (1) cardiovascular responses to L-cysteine microinjected into the rostral ventrolateral medulla (RVLM), where a group of neurons regulate activities of cardiovascular sympathetic neurons and (2) involvement of ionotropic excitatory amino acid (iEAA) receptors in response. In the RVLM of urethane-anesthetized rats accessed ventrally and identified with pressor responses to L-glutamate (10mM, 34nl), microinjections of L-cysteine increased ABP and HR dose dependently (3-100mM, 34nl). The cardiovascular responses to L-cysteine (30mM) were not attenuated by a prior injection of either antagonist alone, MK801 (20mM, 68nl) for the NMDA type of iEAA receptors, or CNQX (2mM) for the non-NMDA type. However, inhibition of both NMDA and non-NMDA receptors with additional prior injection of either antagonist completely blocked those responses to L-cysteine. The results indicate that L-cysteine has functional cardiovascular action in the RVLM of the anesthetized rat, and the responses to L-cysteine involve both NMDA and non-NMDA receptors albeit in a mutually exclusive parallel fashion. The findings may suggest endogenous roles of L-cysteine indirectly via iEAA receptors in the neuronal network of the RVLM for cardiovascular regulation in physiological and pathological situations.

Correction to Chemoenzymatic Synthesis of New 2,4-syn-Functionalized (S)-Glutamate Analogues and Structure–Activity Relationship Studies at Ionotropic Glutamate Receptors and Excitatory Amino Acid Transporters

Correction to Chemoenzymatic Synthesis of New 2,4-<i>syn</i>-Functionalized (<i>S</i>)-Glutamate Analogues and Structure–Activity Relationship Studies at Ionotropic Glutamate Receptors and Excitatory Amino Acid Transporters

Regulation of arterial pressure by the paraventricular nucleus in conscious rats: interactions among glutamate, GABA, and nitric oxide

The paraventricular nucleus (PVN) of the hypothalamus is an important site for autonomic and neuroendocrine regulation. Experiments in anesthetized animals and in vitro indicate an interaction among gamma-aminobutyric acid (GABA), nitric oxide (NO), and glutamate in the PVN. The cardiovascular role of the PVN and interactions of these neurotransmitters in conscious animals have not been evaluated fully. In chronically instrumented conscious rats, mean arterial pressure (MAP) and heart rate (HR) responses to microinjections (100 nl) in the region of the PVN were tested. Bilateral blockade of ionotropic excitatory amino acid (EAA) receptors (kynurenic acid, Kyn) in the PVN produced small but significant decreases in MAP and HR. GABAA receptor blockade (bicuculline, Bic), and inhibition of NO synthase [(NOS), N-(G)-monomethyl-L-arginine, L-NMMA] each increased MAP and HR. The NO donor sodium nitroprusside (SNP) produced depressor responses that were attenuated by Bic. NOS inhibition potentiated both pressor responses to the selective EAA agonist, N-methyl-D-aspartic acid (NMDA), and depressor responses to Kyn. Increases in MAP and HR due to Bic were blunted by prior blockade of EAA receptors. Thus, pressor responses to GABA blockade require EAA receptors and GABA neurotransmission contributes to NO inhibition. Tonic excitatory effects of glutamate in the PVN are tonically attenuated by NO. These data demonstrate that, in the PVN of conscious rats, GABA, glutamate, and NO interact in a complex fashion to regulate arterial pressure and HR under normal conditions.

Intracisternally Injected L-Proline Activates Hypothalamic Supraoptic, but Not Paraventricular, Vasopressin-Expressing Neurons in Conscious Rats

When injected into specific rat brain regions, the neurotransmitter candidate L-proline produces various cardiovascular changes through ionotropic excitatory amino acid receptors. The present study used an immunohistochemical double-labeling approach to determine whether intracisternally injected L-proline in freely moving rats, which increases blood pressure, activates hypothalamic vasopressin-expressing neurons and ventral medullary tyrosine-hydroxylase- (TH-) containing neurons. Following injection of L-proline, the number of activated hypothalamic neurons that coexpressed vasopressin and c-Fos was much greater in the supraoptic nucleus (SON) than in the paraventricular nucleus (PVN) of rats with increased blood pressure. The number of activated TH-containing neurons was significantly greater following L-proline treatment than following control injections of artificial cerebrospinal fluid (ACSF). These results clearly demonstrate that intracisternally injected L-proline activates hypothalamic supraoptic, but not paraventricular, vasopressin-expressing neurons and medullary TH-containing (A1/C1) neurons in freely moving rats.

Altered tryptophan metabolism in Parkinson's disease: A possible novel therapeutic approach

Parkinson's disease (PD) is one of the most common disabling neurodegenerative diseases. Although several therapeutic approaches are available, there are two major unresolved issues: the lack of proved neuroprotective therapy and the treatment of L-dopa-induced motor complications. In the brain, 90% of the tryptophan is metabolized in the kynurenine pathway. Some of the intermediates, such as quinolinic acid and 3-hydroxy kynurenine, are neurotoxic, while others, such as kynurenic acid, are putative intrinsic neuroprotective compounds, mainly by acting as endogenous antagonists of ionotropic excitatory amino acid receptors. Alterations in the kynurenine pathway have been demonstrated in PD. Preclinical data suggest that intervention in the kynurenine pathway may result in neuroprotection and may alleviate L-dopa-induced dyskinesia. There are two alternative approaches for such intervention: the use of kynurenic acid analogues or pro-drugs, or modulation of the activities of the intrinsic enzymes of the pathway.

Is visceral sympathoexcitation to heat stress dependent on activation of ionotropic excitatory amino acid receptors in the rostral ventrolateral medulla?

Acute heat stress activates visceral sympathetic nerve discharge (SND) in young rats, and the functional integrity of the rostral ventrolateral medulla (RVLM) is required for sustaining visceral sympathoexcitation during peak increases in internal body temperature (T(c)). However, RVLM mechanisms mediating SND activation to hyperthermia remain unknown. In the present study, we investigated the role of RVLM ionotropic excitatory amino acid receptors in mediating visceral SND activation to heat stress in anesthetized, young rats. The effects of bilateral RVLM kynurenic acid (Kyn; 2.7 and 5.4 nmol), saline, or muscimol (400-800 pmol) microinjections on renal SND and splenic SND responses to heat stress were determined at peak hyperthermia (T(c) 41.5°C), during progressive hyperthermia (T(c) 40°C), and at the initiation of heating (T(c) increased from 38 to 38.5°C). RVLM Kyn microinjections did not reduce renal and splenic SND recorded during progressive or peak hyperthermia and did not attenuate SND activation at the initiation of heating. In fact, renal and splenic SND tended to be or were significantly increased following RVLM Kyn microinjections at the initiation of heating and during hyperthermia (40 and 41.5°C). RVLM muscimol microinjections at 39, 40, and 41.5°C resulted in immediate reductions in SND. These data indicate that RVLM ionotropic glutamate receptors are required for mediating visceral sympathoexcitation to acute heating and suggest that acute heating activates an RVLM ionotropic excitatory amino acid receptor dependent inhibitory input, which reduces the level of visceral SND to heating.

Human kynurenine aminotransferase II – reactivity with substrates and inhibitors

Kynurenine aminotransferase (KAT) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the conversion of kynurenine, an intermediate of the tryptophan degradation pathway, into kynurenic acid, an endogenous antagonist of ionotropic excitatory amino acid receptors in the central nervous system. KATII is the prevalent isoform in mammalian brain and a drug target for the treatment of schizophrenia. We have carried out a spectroscopic and functional characterization of both the human wild-type KATII and a variant carrying the active site mutation Tyr142→Phe. The transamination and the β-lytic activity of KATII towards the substrates kynurenine and α-aminoadipate, the substrate analog β-chloroalanine and the inhibitors (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxobutanoic acid and cysteine sulfinate were investigated with both conventional assays and a novel continuous spectrophotometric assay. Furthermore, for high-throughput KATII inhibitor screenings, an endpoint assay suitable for 96-well plates was also developed and tested. The availability of these assays and spectroscopic analyses demonstrated that (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxobutanoic acid and cysteine sulfinate, reported to be KATII inhibitors, are poor substrates that undergo slow transamination.

ChemInform Abstract: Ionotropic Excitatory Amino Acid Receptor Ligands. Synthesis and Pharmacology of a New Amino Acid AMPA Antagonist.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Pregnancy decreases GABAergic inhibition of the hypothalamic paraventricular nucleus

Depressor responses to peripheral or central infusion of Angiotensin II type 1 (AT 1) receptor antagonists (AT 1X) are greater in pregnant (P) compared to nonpregnant (NP) animals. AT 1 and ionotropic excitatory amino acid (EAA) receptors contribute to pressor responses to GABA A receptor blockade with bicuculline (Bic) in the paraventricular nucleus (PVN) of male rats. Therefore, we hypothesized that GABAergic inhibition is decreased and AT 1 receptors play a greater excitatory role in the PVN of P versus NP rats. Unilateral microinjection of Bic was performed before (Bic 1), after AT 1X (Bic 2), and after AT 1X + EAA blockade (kynurenate, Kyn) (Bic 3) in the PVN. Increases in mean arterial pressure (MAP: NP = 20 ± 2; P = 12 ± 2 mmHg), heart rate (HR: NP = 57 ± 6; P = 19 ± 6 beats/min) and renal sympathetic nerve activity (RSNA: NP = 70 ± 9; P = 33 ± 7%) due to Bic (Bic 1) were attenuated in P rats. Responses to AT 1X and Kyn alone were insignificant in both groups. In NP rats, AT 1X attenuated (+ 12 ± 4 mmHg), and AT 1X + Kyn further decreased the pressor response to Bic in the PVN (+ 6 ± 2 mmHg). In P rats AT 1X reduced the pressor response to Bic (+ 5 ± 1 mm Hg), and Kyn had no additional effect (+ 3 ± 1 mmHg). Effects of PVN Bic to alter the autospectra of RSNA were suppressed by prior AT 1X and Kyn in both groups. Thus, tonic GABAergic inhibition is decreased and the contribution of AT 1 receptors in the PVN may be greater in P rats.

S59-02 Role of tryptophan-kynurenine pathway in depression: Psychopathological aspect

It was reported that cytokines such as IFN-γ reduce the synthesis of 5-HT by stimulating the activity of indoleamine 2,3 dioxygenase (IDO) enzyme which degrades tryptophan to kynurenine. Kynurenine is further metabolized to kynurenic acid (KYNA), 3-hydroxykynurenine (3OHK) and quinolinic acid (QA) by kynurenine aminotransferase (KAT), kynurenine 3-monooxygenase (KMO) and kynureninase. Both KMO and kynureninase are also shown to be activated by IFNγ. The 3OHK is neurotoxic apoptotic while QA is the excitotoxic N-methyl-D-aspartate (NMDA) receptor agonist. Conversely KYNA is an antagonist of all three ionotropic excitatory amino acid receptors and considered neuroprotective. In the brain, tryptophan catabolism occurs in the astrocytes and. The astrocytes are shown to produce mainly KYNA whereas microglia and macrophages produced mainly 3OHK and QA. The astrocytes have been demonstrated to metabolise the QA produced by the neighbouring microglia.Tryptophan breakdown has been found to be increased but KYNA, the neuroprotective metabolite is decreased in both blood and cerebrospinal fluid of the patients with major depression compared to healthy controls. Moreover, the ratio between KYNA and 3OHK showed significant correlation with response to treatment. These findings lead to the hypothesis an imbalance neuroprotection-neurodegener-ation in terms of kynurenine metabolites and their immunological and biochemical interactions in the brain might further induce the apoptosis of the neuroprotective astrocytes and the vulnerability to stress is thereby enhanced.

Cerebrolysin lowers kynurenic acid formation — An in vitro study

The therapeutic effect of Cerebrolysin in the treatment of dementia and brain injury has been proposed because of neurotrophic properties of this compound. Since an increased kynurenine metabolism has been documented in several brain pathologies including dementia the aim of the present study was to investigate the biochemical properties of Cerebrolysin with respect to kynurenic acid (KYNA) formation in an in vitro study. KYNA is an endogenous metabolite of the kynurenine pathway of tryptophan degradation and is an antagonist of the glutamate ionotropic excitatory amino acid and of the nicotine cholinergic receptors. The activities of the KYNA synthesizing enzymes kynurenine aminotransferases I, II and III (KAT I, KAT II and KAT III) in rat liver, and rat and human brain homogenates were analysed in the presence of Cerebrolysin. KAT I, II and III activities were measured using a radio-enzymatic method in the presence of 1 mM pyruvate and 100 µM [H 3] l-kynurenine. Cerebrolysin, dose-dependently and significantly reduced KAT I, KAT II and KAT III activities of rat liver homogenate. Furthermore, Cerebrolysin exerted a dose-dependent inhibition of rat and human brain KAT I, KAT II and KAT III activities, too. The inhibitory effect of Cerebrolysin was more pronounced for KAT I than for KAT II and KAT III. The present study for the first time demonstrates the ability of Cerebrolysin to lower KYNA formation in rat liver as well as in rat and human brain homogenates. We propose Cerebrolysin as a compound susceptible of therapeutic exploitation in some disorders associated with elevated KYNA metabolism in the brain and/or other tissues. We suggest that the anti-dementia effect of Cerebrolysin observed in Alzheimer patients could be in part due to Cerebrolysin induced reduction of KYNA levels, thus modulating the cholinergic and glutamatergic neurotransmissions.

Brainstem sites for regulation of gill ventilation mapped by microinjection of DLH in the pre‐metamorphic bullfrog (Lithobates catesbeiana)

This study examined the role of ionotropic excitatory amino acid (EAA) receptors for regulating gill‐related respiratory rhythm in the bullfrog brainstem. Our objective was to ‘map’ the sites responsible for generating rhythmic gill burst activity by microinjection of the EAA agonist DL‐Homocysteic Acid (DLH). We used an in vitro brainstem preparation from pre‐metamorphic tadpoles (N=8; T‐K stages 8–16) that was superfused with oxygenated artificial CSF (20–22 °C) and generated spontaneous respiratory motor output measured by suction electrodes attached to cranial nerves (CN) V, X and XII. Pressure injections of small volumes (2–4 nL) of DLH in glass micropipettes were made in 7 locations from the rostral margin of CN V to the caudal margin of CN XII, lateral to the midline and at depths from 100 to 400 microns. DLH produced both inhibitory and excitatory responses. Inhibitory responses were largely confined to an area delineated by the extent of CN VII whereas excitatory responses, consisting of increases in respiratory frequency and burst amplitude, occurred in areas defined by the caudal margin of CN VIII to the rostral margin of CN XII and at most depths examined. These results indicate that EAAs have a widespread influence on gill burst activity in pre‐metamorphic bullfrogs that is not confined to a localized area of the brainstem. Supported by NIH SO6 GM48135.

Activation of ionotropic excitatory amino acid (EAA) receptors modulates both eupnea and gasping in the arterially‐perfused adult rat preparation

Previous work from our laboratory in an arterially‐perfused adult rat preparation has shown that blockade of ionotropic EAA receptors reduces or abolishes basal phrenic nerve discharge and markedly enhances inspiratory neural network complexity. Further, we have demonstrated that during EAA receptor blockade, gasping can still be elicited. The effect of activation of ionotropic glutamate receptors on phrenic motor discharge requires clarification. We examined the effects of perfusion with the glutamate analog DLH on phrenic burst timing and inspiratory neural network complexity in the arterially‐perfused adult rat preparation. DLH (500 μM, n=8) transiently increased phrenic burst frequency by 41% (P=0.005) mediated by decreasing the duration between bursts by 55% (p&lt;0.001), followed by a 36% increased stabilization of burst frequency (P=0.074). Post‐inspiratory activity was also increased. DLH decreased the duration of the peripheral chemoreflex from 23.8±1.7 to 13.2±1.4 s (P&lt;0.001), the duration of ischemia‐induced gasping from 58.6±5.3 to 33.4±2.3 s (P&lt;0.001), and the onset latency to gasping by 79% (P&lt;0.001). These findings support a role for ionotropic EAA neurotransmission in establishing the temporal dynamics of basal phrenic nerve discharge and suggest that it also has a role in determining the onset and duration of the peripheral chemoreflex and gasping. Supported by NS045321 and NS049310

Central efferent pathways mediating skin cooling-evoked sympathetic thermogenesis in brown adipose tissue

Control of thermoregulatory effectors by the autonomic nervous system is a critical component of rapid cold-defense responses, which are triggered by thermal information from the skin. However, the central autonomic mechanism driving thermoregulatory effector responses to skin thermal signals remains to be determined. Here, we examined the involvement of several autonomic brain regions in sympathetic thermogenic responses in brown adipose tissue (BAT) to skin cooling in urethane-chloralose-anesthetized rats by monitoring thermogenic [BAT sympathetic nerve activity (SNA) and BAT temperature], metabolic (expired CO(2)), and cardiovascular (arterial pressure and heart rate) parameters. Acute skin cooling, which did not reduce either rectal (core) or brain temperature, evoked increases in BAT SNA, BAT temperature, expired CO(2), and heart rate. Skin cooling-evoked thermogenic, metabolic, and heart rate responses were inhibited by bilateral microinjections of bicuculline (GABA(A) receptor antagonist) into the preoptic area (POA), by bilateral microinjections of muscimol (GABA(A) receptor agonist) into the dorsomedial hypothalamic nucleus (DMH), or by microinjection of muscimol, glycine, 8-OH-DPAT (5-HT(1A) receptor agonist), or kynurenate (nonselective antagonist for ionotropic excitatory amino acid receptors) into the rostral raphe pallidus nucleus (rRPa) but not by bilateral muscimol injections into the lateral/dorsolateral part or ventrolateral part of the caudal periaqueductal gray. These results implicate the POA, DMH, and rRPa in the central efferent pathways for thermogenic, metabolic, and cardiac responses to skin cooling, and suggest that these pathways can be modulated by serotonergic inputs to the medullary raphe.