Early-life inflammation with LPS delays fear extinction in adult rodents.

A large body of evidence has been brought forward connecting developmental immune activation to abnormal fear and anxiety levels. Anxiety disorders have extremely high lifetime prevalence, yet susceptibility factors that contribute to their emergence are poorly understood. In this research we investigated whether an inflammatory insult early in life can alter the response to fear conditioning in adulthood. Fear learning and extinction are important and adaptive behaviors, mediated largely by the amygdala and its interconnectivity with cortico-limbic circuits. Male and female rat pups were given LPS (100µg/kg i.p.) or saline at postnatal day 14; LPS activated cFos expression in the central amygdala 2.5h after exposure, but not the basal or lateral nuclei. When tested in adulthood, acquisition of an auditory cued or contextual learned fear memory was largely unaffected as was the extinction of fear to a conditioned context. However, we detected a deficit in auditory fear extinction in male and female rats that experienced early-life inflammation, such that there is a significant delay in fear extinction processes resulting in more sustained fear behaviors in response to a conditioned cue. This response was specific to extinction training and did not persist into extinction recall. The effect could not be explained by differences in pain threshold (unaltered) or in baseline anxiety, which was elevated in adolescent females only and unaltered in adolescent males and adult males and females. This research provides further evidence for the involvement of the immune system during development in the shaping of fear and anxiety related behaviors.

Deficient adolescent social behavior following early-life inflammation is ameliorated by augmentation of anandamide signaling.

Early-life inflammation has been shown to exert profound effects on brain development and behavior, including altered emotional behavior, stress responsivity and neurochemical/neuropeptide receptor expression and function. The current study extends this research by examining the impact of inflammation, triggered with the bacterial compound lipopolysaccharide (LPS) on postnatal day (P) 14, on social behavior during adolescence. We investigated the role that the endocannabinoid (eCB) system plays in sociability after early-life LPS. To test this, multiple cohorts of Sprague Dawley rats were injected with LPS on P14. In adolescence, rats were subjected to behavioral testing in a reciprocal social interaction paradigm as well as the open field. We quantified eCB levels in the amygdala of P14 and adolescent animals (anandamide and 2-arachidonoylglycerol) as well as adolescent amygdaloid cannabinoid receptor 1 (CB1) binding site density and the hydrolytic activity of the enzyme fatty acid amide hydrolase (FAAH), which metabolizes the eCB anandamide. Additionally, we examined the impact of FAAH inhibition on alterations in social behavior. Our results indicate that P14 LPS decreases adolescent social behavior (play and social non-play) in males and females at P40. This behavioral alteration is accompanied by decreased CB1 binding, increased anandamide levels and increased FAAH activity. Oral administration of the FAAH inhibitor PF-04457845 (1mg/kg) prior to the social interaction task normalizes LPS-induced alterations in social behavior, while not affecting social behavior in the control group. Infusion of 10ng PF-04457845 into the basolateral amygdala normalized social behavior in LPS injected females. These data suggest that alterations in eCB signaling following postnatal inflammation contribute to impairments in social behavior during adolescence and that inhibition of FAAH could be a novel target for disorders involving social deficits such as social anxiety disorders or autism.

Fever and sickness behavior: Friend or foe?

Fever has been recognized as an important symptom of disease since ancient times. For many years, fever was treated as a putative life-threatening phenomenon. More recently, it has been recognized as an important part of the body's defense mechanisms; indeed at times it has even been used as a therapeutic agent. The knowledge of the functional role of the central nervous system in the genesis of fever has greatly improved over the last decade. It is clear that the febrile process, which develops in the sick individual, is just one of many brain-controlled sickness symptoms. Not only will the sick individual appear "feverish" but they may also display a range of behavioral changes, such as anorexia, fatigue, loss of interest in usual daily activities, social withdrawal, listlessness or malaise, hyperalgesia, sleep disturbances and cognitive dysfunction, collectively termed "sickness behavior". In this review we consider the issue of whether fever and sickness behaviors are friend or foe during: a critical illness, the common cold or influenza, in pregnancy and in the newborn. Deciding whether these sickness responses are beneficial or harmful will very much shape our approach to the use of antipyretics during illness.

Neonatal programming of innate immune function.

The early life environment can be crucial in influencing the development of an animal's long-term physiology. There is now much evidence to suggest that perinatal challenges to an animal's immune system will result in changes in adult rat behavior, physiology, and molecular pathways following a single inflammatory event during development caused by the bacterial endotoxin lipopolysaccharide (LPS). In particular, it is now apparent that neonatal LPS administration can influence the adult neuroimmune response to a second LPS challenge through hypothalamic-pituitary-adrenal axis modifications, some of which are caused by alterations in peripheral prostaglandin synthesis. These pronounced changes are accompanied by a variety of alterations in a number of disparate aspects of endocrine physiology, with significant implications for the health and well-being of the adult animal. In this review, we discuss the newly elucidated mechanisms by which neonatal immune challenge can permanently alter an animal's endocrine and metabolic physiology and the implications this has for various disease states.

Viral-like brain inflammation during development causes increased seizure susceptibility in adult rats.

Viral infections of the CNS and their accompanying inflammation can cause long-term neurological effects, including increased risk for seizures. To examine the effects of CNS inflammation, we infused polyinosinic:polycytidylic acid, intracerebroventricularly to mimic a viral CNS infection in 14 day-old rats. This caused fever and an increase in the pro-inflammatory cytokine, interleukin (IL)-1beta in the brain. As young adults, these animals were more susceptible to lithium-pilocarpine and pentylenetetrazol-induced seizures and showed memory deficits in fear conditioning. Whereas there was no alteration in adult hippocampal cytokine levels, we found a marked increase in NMDA (NR2A and C) and AMPA (GluR1) glutamate receptor subunit mRNA expression. The increase in seizure susceptibility, glutamate receptor subunits, and hippocampal IL-1beta levels were suppressed by neonatal systemic minocycline. Thus, a novel model of viral CNS inflammation reveals pathophysiological relationships between brain cytokines, glutamate receptors, behaviour and seizures, which can be attenuated by anti-inflammatory agents like minocycline.

Thyrotropin-releasing hormone selectively depresses glutamate excitation of cerebral cortical neurons.

The microiontophoretic application of thyrotropin-releasing hormone causes a selective reduction in neuronal excitation evoked by L-glutamate but not by acetylcholine in rat cerebral cortex. Thyrotropin-releasing hormone has no influence on the activity of acetylcholinesterase or on choline uptake and release from cerebral synaptosomes. This evidence for a selective interaction between a centrally acting peptide and an excitatory amino acid neurotransmitter may indicate a specific locus of thyrotropin-releasing hormone action at glutamate-activated receptor sites.

Dendritically released peptides act as retrograde modulators of afferent excitation in the supraoptic nucleus in vitro.

Oxytocin (OXT) and vasopressin (VP) are known to be released from dendrites of magnocellular neurons. Here, we show that these peptides reduced evoked EPSCs by a presynaptic mechanism, an effect blocked by peptide antagonists and mimicked by inhibition of endogenous peptidases. Dendritic release of peptides, elicited with depolarization achieved by high frequency stimulation of afferents or with current injection into an individual neuron, induced short-term synaptic depression similar to that seen following exogenous peptide application and was prevented by peptide antagonists. Thus, dendritically released peptides depress evoked EPSCs in magnocellular neurons by activating presynaptic OXT and/or VP receptors. Such a retrograde modulatory action on afferent excitation may serve as a feedback mechanism to permit peptidergic neurosecretory neurons to autoregulate their own activity.

Neonatal inflammation produces selective behavioural deficits and alters N-methyl-D-aspartate receptor subunit mRNA in the adult rat brain.

Peripheral inflammation causes production of central cytokines that alter transmission at the N-methyl-D-aspartate receptor (NR). During development, NRs are important for synaptic plasticity and network connectivity. We therefore asked if neonatal inflammation would alter expression of NRs in the brain and behavioural performance in adulthood. We gave lipopolysaccharide (LPS) (100 microg/kg, i.p.) or saline to male rats on postnatal day (P)5, P14, P30 or P77. Subsequently we assessed mRNA levels of the NR1, NR2A, B, C and D subunits in the hippocampus and cortex either acutely (2 h) or in adulthood using real-time reverse transcriptase-polymerase chain reaction. We explored learning and memory behaviours in adult rats using the Morris water maze and contextual fear conditioning paradigms. Hippocampal NR1 mRNA was acutely increased in the P5- and P77-treated rats but was reduced in adults treated with LPS at P5, P30 and P77. P14 LPS-treated rats showed few acute changes but showed pronounced increases in NR2A, B, C and D subunit mRNA later in adulthood. The cortex displayed relatively few acute changes in expression in the neonatal-treated rats; however, it showed robust changes in NR2B, C and D mRNA in all groups given LPS in adulthood. Behavioural deficits were observed specifically in the P5 and P30 LPS-treated groups in the water maze probe trial and fear conditioning tests, consistent with hippocampal NR1 mRNA down-regulation. Thus, a single bout of inflammation during development can programme specific and persistent differences in NR mRNA subunit expression in the hippocampus, which could be associated with behavioural and cognitive deficits in adulthood.

Vasopressin autoreceptors and nitric oxide-dependent glutamate release are required for somatodendritic vasopressin release from rat magnocellular neuroendocrine cells responding to osmotic stimuli.

Magnocellular neuroendocrine cells of the supraoptic nucleus (SON) release vasopressin (VP) systemically and locally during osmotic challenge. Although both central VP and nitric oxide (NO) release appear to reduce osmotically stimulated systemic VP release, it is unknown whether they interact locally in the SON to enhance somatodendritic release of VP, a phenomenon believed to regulate systemic VP release. In this study, we examined the contribution of VP receptor subtypes and NO to local VP release from the rat SON elicited by systemic injection of 3.5 m saline. Treatment of SON punches with VP receptor antagonists decreased osmotically stimulated intranuclear VP release. Similarly, blockade of NO production, or addition of NO scavengers, reduced stimulated VP, glutamate, and aspartate release, suggesting that local NO production and activity are critical for osmotically induced intranuclear VP and excitatory amino acid release. An increase in endogenous NO release from SON punches in response to hyperosmolality was confirmed by enzymatic NO assay. Consistent with enhanced glutamate and VP release from stimulated rat SON punches, the ionotropic glutamate receptor blocker kynurenate decreased stimulated local VP release without affecting NO release. These data suggest that NO enhances local VP release in part by facilitating local release of glutamate/aspartate and that glutamate receptor activity is required for the stimulation of local VP release by osmotic challenge. Collectively, these results suggest that local VP receptors, NO, and glutamatergic signaling mediate the amplification of intranuclear VP release during hyperosmolality and may contribute to efficient, but not exhaustive, systemic release of VP during osmoregulatory challenge.

A novel role for endogenous pituitary adenylate cyclase activating polypeptide in the magnocellular neuroendocrine system.

Central release of vasopressin (VP) by the magnocellular neuroendocrine cells (MNCs) responsible for systemic VP release is believed to be important in modulating the activity of these neurons during dehydration. Central VP release from MNC somata and dendrites is stimulated by both dehydration and pituitary adenylate cyclase activating polypeptide (PACAP). Although PACAP is expressed in MNCs, its potential role in the magnocellular response to dehydration is unexplored. The current study demonstrates that prolonged dehydration increases immunoreactivity for PACAP-27, PACAP-38, and the type I PACAP receptor in the supraoptic nucleus (SON) of the rat. In addition, PACAP stimulates local VP release in the euhydrated rat SON in vitro, and this effect is reduced by the PACAP receptor antagonist PAC(6-27) (100 nm), suggesting the participation of PACAP receptors. Concomitant with its effects on local VP release, PACAP also reduces basal glutamate and aspartate release in the euhydrated rat SON. Furthermore, somatodendritic VP release elicited by acute dehydration is blocked by PAC(6-27), suggesting that endogenous PACAP participates in this response. Consistent with this, RIA revealed that local PACAP-38 release within the SON is significantly elevated during acute dehydration. These results suggest that prolonged activation of hypothalamic MNCs is accompanied by up-regulation of PACAP and the type I PACAP receptor in these cells and that somatodendritic VP release in response to acute dehydration is mediated by activation of PACAP receptors by endogenous PACAP released within the SON. A potential role for PACAP in promoting efficient, but not exhaustive, systemic release of VP from MNCs during physiological challenge is discussed.

Attenuation of fever at near term: is interleukin-6-STAT3 signalling altered?

Pregnant rats in late gestation show a reduced fever response after stimulation with lipopolysaccharide (LPS). This can result from either an increased action of endogenous antipyretics or a reduction in the production or action of endogenous pyrogens. Nonpregnant rats given LPS release interleukin (IL)-6, which causes nuclear translocation of the signal transducer and activator of transcription 3 (STAT3) in the vascular organ of the lamina terminalis (OVLT), followed by a significant increase in core body temperature. The present study investigated whether the reduced fever response in near-term pregnant rats is associated with a reduced nuclear STAT3 response. Rats at gestation day 15 (G15), gestation day 21 (G21, near term) and at lactation day 5 (L5) were injected with LPS (50 microg/kg, i.p.) or vehicle. Only near-term pregnant rats responded with an attenuated body temperature during the fever response. Immunohistological analysis indicated no significant difference in nuclear STAT3 in the OVLT of the different animal groups 2 h after LPS. Measurement of total and phosphorylated STAT3 protein in the OVLT with semiquantitative western blot revealed no significant differences of this protein among these immune challenged animal groups. IL-6 concentrations were also similar at G15, G21 and L5 2 h after injection of LPS. These results lead to the conclusion that the attenuation of the fever response at near-term pregnancy is not associated with a reduced amount of nuclear STAT3 in the OVLT, indicating a maintained IL-6-STAT3 signalling pathway in the OVLT.

Disruption of the blood-brain barrier during TNBS colitis.

Well-documented central nervous system changes during colitis suggest possible alterations of blood-brain barrier (BBB) permeability, yet the integrity of the BBB has not been fully evaluated in experimental colitis. Our aim was to investigate whether trinitrobenzene sulphonic acid (TNBS) colitis was associated with an increase in the permeability of the BBB. Sprague-Dawley rats were given an intracolonic injection of saline or TNBS and studied 1, 2, 3, 7 and 21 days after treatment. The extravasation of endogenous immunoglobulin G, a large molecule, was not altered at any time after TNBS treatment. In contrast, significant increases in the BBB leakage of sodium fluorescein, a much smaller molecule, were observed 1 and 2 days after the induction of colitis, in and around the circumventricular organs; the organum vasculosum of the lamina terminalis, subfornical organ and median eminence of the hypothalamus. TNBS-treated rats also exhibited sodium fluorescein leakage in focal areas in the brain parenchyma. The expression of endothelial barrier antigen, a protein associated with the BBB, was reduced about 60% 48 h after the induction of colitis. This returned to control values by 3 weeks, when colitis had largely subsided. In conclusion, experimental colitis transiently increased permeability of the brain to small molecules through a mild disruption of the BBB.

Neuroimmune response to endogenous and exogenous pyrogens is differently modulated by sex steroids.

The objective of this study was to explore whether and how ovarian hormones interact with the febrile response to pyrogens. Estrogen and progesterone treatment of ovariectomized rats was associated with a reduction in lipopolysaccharide (LPS)-induced fever, compared with ovariectomized controls. LPS-fever reduction was accompanied by reduced levels of the inducible cyclooxygenase-2 (COX-2) protein expression in the hypothalamus as well as reduced plasma levels of IL-1beta. The amount of LPS-induced IL-6 in the plasma was not affected by ovarian hormone replacement. In contrast, hypothalamic COX-2 expression in response to intraperitoneal injection of IL-1beta was potentiated by the ovarian hormone replacement. IL-1beta induced a moderate increase in plasma levels of IL-6 that was suppressed by ovarian hormone replacement. These data suggest that ovarian hormone replacement attenuated the proinflammatory response to LPS by suppressing the LPS-induced IL-1beta production and COX-2 expression in the hypothalamus. The markedly different action of ovarian hormones on IL-1beta and LPS effects suggests that this sex hormone modulation of the immune response is a function of the nature of infection and provides further evidence that LPS actions are different from those of IL-1beta.

Central effects of arginine vasopressin on blood pressure in rats.

In anesthetized male Sprague-Dawley rats, intraventricular injection of 25-5000 pmol AVP caused short-latency, dose-related increases in blood pressure. Blood pressure increases occurred before any changes in urine flow were observed, suggesting that the AVP was acting centrally. Similar increases in blood pressure were observed after central AVP injection into homozygous Brattleboro rats which lack AVP, indicating these effects were not due to release of pituitary AVP stores. These results raise the possibility that endogenous AVP plays a role in the central control of blood pressure.

GABA(B) receptors modulate short-term potentiation of spontaneous excitatory postsynaptic currents in the rat supraoptic nucleus in vitro.

High-frequency stimulation of afferents to the supraoptic nucleus (SON) results in a robust increase in the frequency and amplitude of pharmacologically isolated, tetrodotoxin-resistant, miniature excitatory postsynaptic currents (mEPSCs) lasting for 5-20 min. This increase in mEPSC frequency, termed short-term potentiation (STP), is tightly coupled to increases in action potential firing in magnocellular neurons (MCNs) suggesting a functional role for STP. gamma-Aminobutyric acid (GABA), acting selectively on GABA(B) receptors, has been shown to modulate action potential-dependent EPSCs, as well as mEPSCs in this nucleus. In this study, we examined the role of GABA in STP. Using in vitro hypothalamic slices containing the SON and the nystatin perforated-patch recording technique to record from MCNs, we tested the hypothesis that GABA modulates STP. Baclofen, a GABA(B) receptor agonist, caused a reversible decrease in the frequency of mEPSCs as well as a reduction in the magnitude and duration of STP. GABA(B) receptor antagonists blocked the baclofen-induced decrease in mEPSC frequency and reduction in STP. In addition, the antagonists by themselves increased basal mEPSC frequency while prolonging the duration of STP in most cells. By contrast, picrotoxin, a GABA(A) chloride channel blocker, had no effect on STP.These findings indicate that GABA is tonically present in the SON and its action at the GABA(B) receptor may determine the magnitude and duration of STP.

Responses of electrophysiologically identified rat paraventricular neurons to cholecystokinin and other stimuli.

Extracellular recordings were carried out in the paraventricular nucleus of halothane-anesthetized male rats. Responses of neurons identified by antidromic criteria with projections to the nucleus tractus solitarius or to the ventral lateral medulla were compared to those of neurohypophysial neurons following alterations in blood pressure and osmolarity, hemorrhage and after intravenous injection of cholecystokinin. Neurohypophysial neurons displayed the well-described responses to blood pressure for putative vasopressin neurons and increases in excitability after cholecystokinin for putative oxytocin neurons. Twenty per cent of the ventral lateral medulla-projecting neurons were responsive to cardiovascular perturbations, with these displaying reduced activity after either decreases or increases in blood pressure. None of nine neurons projecting to the ventral lateral medulla responded to i.v. cholecystokinin. Two of 20 nucleus tractus solitarius-projecting neurons showed reduced activity after cholecystokinin and none increased their firing rate. Nitroprusside-induced hypotension was associated with reduced activity in 10% of this population. Three neurons displayed axon projections to both pituitary and medulla; two of these which projected to the nucleus tractus solitarius were activated by cholecystokinin. We conclude that some of the paraventricular nucleus neurons projecting to the medulla respond to recognized cardiovascular stimuli for neurohypophysial neurons, but neurons in these populations are generally unresponsive to cholecystokinin. The former group of neurons may act to coordinate autonomic and endocrine responses to cardiovascular perturbation; however, there may be other stimuli, such as cholecystokinin, which act only on one of the populations of paraventricular nucleus neurons. Furthermore, many neurons in the descending pathways may respond to stimuli not presently associated with activation of magnocellular neurons.

Changes in arterial blood pressure alter activity of electrophysiologically identified single units of the bed nucleus of the stria terminalis.

The bed nucleus of the stria terminalis may play a role in cardiovascular function by way of its connectivity to the diagonal band of Broca/ventral septal area. The present study sought to determine whether changes in systemic blood pressure affect the electrical activity of single units within the bed nucleus of the stria terminalis. Extracellular voltage recordings from neurons in the bed nucleus were performed in urethane-anaesthetized rats catheterized for arterial blood pressure measurements and for the intravenous administration of pressor and depressor drugs. Afferent or efferent connectivity of each recorded neuron was determined following electrical stimulation of nearby nuclei with and without known barosensitive regions. Of neurons demonstrating efferent connectivity (antidromically evoked potentials) with the diagonal band of Broca/ventral septal area or habenular nuclei, 24 and 20%, respectively, responded to changes in blood pressure with either increases or decreases in firing frequency. Paraventricular nucleus-projecting neurons were not affected by alterations in arterial blood pressure. Orthodromic potentials (inhibitory and/or excitatory) in the bed nucleus were also observed following stimulation of these nearby nuclei. Of these orthodromically activated neurons, changes in arterial pressure affected 31% of neurons receiving input from the diagonal band of Broca/ventral septal area, 33% of neurons with connectivity to the habenular nuclei and 60% of neurons with connectivity to the paraventricular nucleus. These data show that the bed nucleus of the stria terminalis contains a sub-population of cells that are sensitive to deviations in resting arterial pressure and that these cells receive synaptic modulation from several limbic/forebrain sources. Furthermore, the results are consistent with a role for the bed nucleus in the control of cardiovascular function and as a relay nucleus for modified baroreceptor input toward the diagonal band of Broca/ventral septal area.

Acute, sequence-specific effects of oxytocin and vasopressin antisense oligonucleotides on neuronal responses.

Antisense oligodeoxynucleotides (antisense) are short length single strands of DNA with base sequences complementary to a length of messenger RNA of a specific gene. They can be taken up by neurons and hybridize with a complementary messenger RNA to selectively interrupt the expression of a particular gene. We now describe neuropeptide-specific, short-latency (within 2-6 h) effects of antisense infused into the supraoptic nucleus on the responses of rat neurohypophysical neurons, in vivo, to various stimuli. Oxytocin antisense specifically (i) reduced the electrophysiological responses of putative oxytocin, but not vasopressin neurons, (ii) inhibited cholecystokinin-induced and electrically stimulated release of oxytocin from the neurohypophysis, and (iii) reversibly abolished cholecystokinin-induced expression of Fos within the supraoptic nucleus. Vasopressin antisense reduced the excitatory responses of vasopressin neurons, but not of oxytocin neurons. As neuropeptide content within the supraoptic nucleus and neurohypophysis remains unaltered at this time, antisense may induce anticipatory, feed-forward alterations in electrical activity in addition to any possible effects on peptide synthesis.

A dopaminergic inhibitory postsynaptic potential mediated by an increased potassium conductance.

Intracellular recordings from intact pituitary melanotrophs show that, in the same cell, inhibitory postsynaptic potentials resulting from either pituitary stalk stimulation or exogenous dopamine are abolished by D2 receptor antagonists, display identical conductance changes, are reversed in polarity at the same membrane potential and are sensitive to pertussis toxin pretreatment. The reversal potential of the inhibitory postsynaptic potential shows a 65 mV shift with a 10-fold change in external potassium concentration, which is close to that predicted by the Nernst equation. We conclude that activation of this synapse releases dopamine which acts on a D2 receptor to increase potassium conductance via a G-protein-mediated mechanism. This is the first characterization of an inhibitory dopaminergic synapse in the mammalian nervous system.

Lateral septum-medial hypothalamic connections: an electrophysiological study in the rat.

Participation of both the lateral septum and the medial hypothalamus in similar behavioral and homeostatic functions, combined with anatomical data indicating a modest descending lateral septum projection to medial hypothalamic areas, prompted an investigation of the electrophysiology of lateral septum--medial hypothalamic connections in pentobarbital-anesthetized male Sprague-Dawley rats. Field potentials evoked by lateral septum stimulation were prominent within the hypothalamic ventromedial nucleus: their distribution closely overlapped with amygdala-evoked field potentials. Lateral septum stimulation evoked orthodromic responses from 67% of 800 medial hypothalamic and preoptic neurons (55.5% excitatory, latency 17.5 +/- 0.5 ms; 11.5% inhibitory, latency 19.1 +/- 1.4 ms), including 15 of 31 tuberoinfundibular neurons activated antidromically from the median eminence, 37.4% of cells responded orthodromically to both lateral septum and amygdala stimulation, whereas only 5.8% of cells demonstrated orthodromic responses to both lateral septum and midbrain periaqueductal gray stimulation. These observations suggest that lateral septum neurons influence the excitability of many medial preoptic and hypothalamic neurons, and indicate a convergence of lateral septum and amygdala influences onto 37% of medial hypothalamic cells. In the rat, the lateral septum's influence on adenohypophyseal hormone secretion appears to be mediated indirectly, i.e. through monosynaptic connections with medial hypothalamic tuberoinfundibular neurons.