Sustained Activation of the Anterior Thalamic Neurons with Low Doses of Kainic Acid Boosts Hippocampal Neurogenesis.

Adult hippocampal neurogenesis is prone to modulation by several intrinsic and extrinsic factors. The anterior nucleus (AN) of the thalamus has extensive connections with the hippocampus, and stimulation of this region may play a role in altering neurogenesis. We have previously shown that electrical stimulation of the AN can substantially boost hippocampal neurogenesis in adult rats. Here, we performed selective unilateral chemical excitation of the cell bodies of the AN as it offers a more specific and sustained stimulation when compared to electrical stimulation. Our aim is to investigate the long-term effects of KA stimulation of the AN on baseline hippocampal proliferation of neural stem cells and neurogenesis. Continuous micro-perfusion of very low doses of kainic acid (KA) was administered into the right AN for seven days. Afterwards, adult male rats received 5'-bromo-2'-deoxyuridine (BrdU) injections (200 mg/kg, i.p) and were euthanized at either one week or four weeks post micro-perfusion. Open field and Y-maze tests were performed before euthanasia. The KA stimulation of the AN evoked sustained hippocampal neurogenesis that was associated with improved spatial memory in the Y-maze test. Administering dexamethasone prior to and simultaneously with the KA stimulation decreased both the hippocampal neurogenesis and the improved spatial recognition memory previously seen in the Y-maze test. These results suggest that hippocampal neurogenesis may be a downstream effect of stimulation in general, and of excitation of the cell bodies of the AN in particular, and that stimulation of that area improves spatial memory in rats.

Long-term stimulation of the anteromedial thalamus increases hippocampal neurogenesis and spatial reference memory in adult rats.

Deep brain stimulation (DBS) has shown positive clinical results in neurodegenerative diseases. Previous work from our group showed that a single session of DBS to the anteromedial thalamic nucleus (AMN) in awake rats, increased proliferation of stem/progenitor cells in the dentate gyrus (DG) of the hippocampus. We thought to examine the effect of single versus multiple sessions of DBS to the AMN in modulating adult hippocampal neurogenesis. Rats received unilateral single session, multiple sessions or no electrical stimulation (sham) in the right AMN. Rats received 5'-bromo-2'-deoxyuridine (BrdU) injections and were followed over a period of 1 week or 4 weeks. Single session of electrical stimulation induced a 1.9-fold increase in the number of proliferating BrdU positive cells after one week from stimulation and a 1.8-fold increase at four weeks post stimulation, both in the ipsilateral DG. As for multiple sessions of stimulation, they induced a 3- fold increase that extended to the contralateral DG after 4 weeks from stimulation. Spatial reference memory was tested in the Y-maze test by examining novel arm exploration. Both single and multiple sessions of stimulation prompted an increase in novel arm exploration at week 4, while only the multiple sessions of stimulation had this effect starting from week 1. This study demonstrates that sustained activation of the AMN boosts neurogenesis and improves spatial reference memory.

Intranigral Injection of Endotoxin Suppresses Proliferation of Hippocampal Progenitor Cells.

Brain inflammation can result in functional disorders observed in several neurodegenerative diseases and that can be also associated with reduced neurogenesis. In this study, we investigate the effect of mild inflammation, induced by unilateral injection of Endotoxin (ET) in the substantia nigra (SN)/Ventral Tegmental Area, on the proliferation and survival of stem/progenitor cells in the dentate gyrus (DG) of the hippocampus. Adult female rats received unilateral injection of ET (2 µg/2 µl saline) or sterile saline (2 µl) in the right SN followed by 5'-Bromo-2'-deoxyuridine (BrdU) injections (66 mg/kg/injection). Intranigral ET injection induced bilateral decrease in the number of newly born BrdU positive cells in the DG. This effect was paralleled by a significant decrease in the exploratory behavior of rats, as assessed by the Y-maze novel arm exploration task. ET also induced a transient decrease in the number of tyrosine hydroxylase-positive cells in the injected SN, impaired motor behavior, and caused microglial activation in the SN. This study provides an experimental simulation of the remote effects of moderate and reversible neuroinflammation resulting in impaired communication between midbrain dopaminergic neurons and the hippocampus.

Contribution of capsaicin-sensitive innervation to the continuous eruption of the rat mandibular incisors.

A major component of tooth innervation is made of capsaicin-sensitive primary afferents (CSPA). These fibers play a key role in tooth pain and inflammation; little is known, however, about the role of CSPA in tooth eruption. The aim of this study was to examine the role of the capsaicin-sensitive afferents in the process of eruption of intact rat incisors. CSPA fibers in several rat groups, were subjected to one of the following experimental procedures: systemic chemical ablation, systemic ablation followed by chemical sympathectomy and localized activation. The observed effects on incisor eruption were compared to those made on controls. The total amount of eruption in control/naïve rats, measured over a total period of 144 h, was 3.18 ± 0.07 mm and decreased to 2.43 ± 0.08 mm (n = 7; p < 0.001) following systemic ablation of CSPA. Further decrease to 2.24 ± 0.08 mm (n = 7; p < 0.001) was noticed when chemical sympathectomy was added to CSPA ablation. The average rate of eruption was 1.7 ± 0.25 mm following CSPA activation, compared to an average of 0.8 ± 0.07 mm for controls (n = 7; p < 0.001). Capsaicin sensitive fibers play an important role in tooth homeostasis, and intact neural supply is required for tooth growth under normal conditions.

Nitrous Oxide Induces Prominent Cell Proliferation in Adult Rat Hippocampal Dentate Gyrus.

The identification of distinct and more efficacious antidepressant treatments is highly needed. Nitrous oxide (N2O) is an N-methyl-D-aspartic acid (NMDA) antagonist that has been reported to exhibit antidepressant effects in treatment-resistant depression (TRD) patients. Yet, no studies have investigated the effects of sub-anesthetic dosages of N2O on hippocampal cell proliferation and neurogenesis in adult brain rats. In our study, adult male Sprague-Dawley rats were exposed to single or multiple exposures to mixtures of 70% N2O and 30% oxygen (O2). Sham groups were exposed to 30% O2 and the control groups to atmospheric air. Hippocampal cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation, and BrdU-positive cells were counted in the dentate gyrus (DG) using confocal microscopy. Results showed that while the rates of hippocampal cell proliferation were comparable between the N2O and sham groups at day 1, levels increased by 1.4 folds at day 7 after one session exposure to N2O. Multiple N2O exposures significantly increased the rate of hippocampal cell proliferation to two folds. Therefore, sub-anesthetic doses of N2O, similar to ketamine, increase hippocampal cell proliferation, suggesting that there will ultimately be an increase in neurogenesis. Future studies should investigate added N2O exposures and their antidepressant behavioral correlates.

Modulation of incisor eruption in rats by sympathetic efferents.

INTRODUCTION: Intact neural supply is necessary for tooth eruption. Sympathetic denervation accelerates or decelerates the eruption rate depending on the tooth condition (intact or injured). The aim of this study is to reexamine the role of the sympathetic innervation, through the observation of the effects of pre or post ganglionic chemical sympathectomy on the eruption of intact rat incisors. MATERIALS AND METHODS: Different groups of rats were subjected to either ganglionic or peripheral chemical sympathectomy and the observed effects on incisor eruption were compared to those made on intact/sham groups or on rats subjected to inferior alveolar nerve (IAN) lesion. RESULTS: The total amount of eruption in control/naïve rats, measured over a total period of 144 h, was 3 ±â€¯0.15 mm and decreased to 2.57 ±â€¯0.06 mm (n = 8; p < 0.01) or 2.8 ±â€¯0.10 mm (n = 8; p < 0.05) following treatment with guanethidine and hexamethonium, respectively. This amount decreased to 1.8 ±â€¯0.14 mm (p < 0.001 vs. control, n = 7; or p < 0.01 vs. sham, n = 5) in rats subjected to IAN lesion. CONCLUSION: Sympathectomy delayed tooth eruption. Blocking the sympathetic effectors with guanethidine exerted more potent effects than ganglionic block with hexamethonium. Intact sympathetic supply is required for tooth growth under normal conditions.

Predisposing effects of neonatal visceral pain on abuse-related effects of morphine in adult male Sprague Dawley rats.

RATIONALE: Adverse early life experiences are risk factors for drug abuse and addiction. Changes in brain opioid systems have been demonstrated in response to neonatal visceral pain (NVP), but the impact of these changes on abuse-related effects of morphine are unknown. The NVP procedure used models chronic visceral hyperalgesia persisting across development. OBJECTIVES: Intravenous self-administration, drug discrimination, and locomotor activity were used to compare the abuse-related effects of morphine in NVP and control rats. METHODS: Rats self-administered 0.3 mg/kg/inj morphine under an FR1 schedule, and dose-effect functions for morphine were then established. Separate rats were trained to discriminate 3.2 mg/kg morphine from saline under an FR20 schedule, and morphine dose-effect functions were then determined in the absence and presence of 0.1 mg/kg naltrexone. A third group of rats was tested with a range of morphine doses in an assay of locomotor activity, then injected daily with 10 mg/kg morphine to assess locomotor sensitization. RESULTS: NVP rats self-administered more morphine than controls at reinforcing doses. Discriminative stimulus effects of morphine were similar between groups, but in the presence of naltrexone, the ED50 for morphine was more than 12× greater in control rats than in NVP animals. Morphine did not stimulate locomotor activity at any tested dose in NVP rats, although significant effects were observed in controls. Finally, significant locomotor sensitization was observed only in NVP rats. CONCLUSIONS: NVP-induced changes in brain opioid systems have persistent pharmacological consequences into adulthood and may increase sensitivity to abuse-related effects of opioids across development.

Altered behavior and digestive outcomes in adult male rats primed with minimal colon pain as neonates.

BACKGROUND: Neonatal colon irritation (CI; pain or inflammation) given for 2 weeks prior to postnatal day 22 (PND22), causes long-lasting functional disorders in rats that can be seen 6 months after the initial insult. This study looked at the effect of varying the frequency and duration of neonatal CI on the rate of growth, digestive outcomes, exploratory activity, and colon and skin sensitivity in adult rats. METHODS: Male Sprague-Dawley rats were given CI using repeated colorectal distension (CRD) at different time intervals and for varying durations starting at PND 8, 10 or 14. Control rats were handled by the investigator without any intracolonic insertion. Further experiments were done on adult rats. Digestive outcomes (food and water consumption, fecal and urinary outputs) were measured using metabolic cages. Exploratory behavior was measured using digital video tracking in an open field. Cutaneous sensitivity was assessed by measuring the responses to mechanical and heat stimuli applied to the shaved abdomen or hind paws. Visceral sensitivity was measured by recording electromyographic responses, under light isoflurane anesthesia, from the external oblique muscles in response to CRD. RESULTS: No significant weight differences were observed between CI and control rats. Exploratory behavior was reduced in rats with neonatal CI compared to control. Digestive outputs and somatic and visceral sensitivity changed between different treatment groups with earlier and more frequent insults yielding a higher deviation from normal. CONCLUSION: The diversity of behavioral and digestive symptoms in these rats parallels the diversity of symptoms in patients with functional gastrointestinal disorders and is consistent with global plastic changes affecting more than one system in the organism.

An improved method for patch clamp recording and calcium imaging of neurons in the intact dorsal root ganglion in rats.

The properties of dorsal root ganglion (DRG) neurons have been mostly investigated in culture of dissociated cells, and it is uncertain whether these cells maintain the electrophysiological properties of the intact DRG neurons. Few attempts have been made to record from DRG neurons in the intact ganglion using the patch clamp technique. In this study, rat DRGs were dissected and incubated for at least 1h at 37 degrees C in collagenase (10mg/ml). We used oblique epi-illumination to visualize DRG neurons and perform patch clamp recordings. All DRG neurons exhibited strong delayed rectifier potassium current and a high threshold for spike generation (-15 mV) that rendered the cells very weakly excitable, generating only one action potential upon strong current injection (>300 pA). It is therefore possible that cultured DRG neurons, commonly used in studies of pain processing, may be hyperexcitable because they acquired "neuropathic" properties due to the injury induced by their dissociation. Electrical stimulation of the attached root produced an antidromic spike in the soma that could be blocked by intracellular hyperpolarization or high frequency stimulation. Imaging intracellular calcium concentration with Oregon Green BAPTA-1 indicates that antidromic stimulation caused a long-lasting increase in intracellular calcium concentration mostly near the cell membrane. This study describes a simple approach to examine the electrophysiological and pharmacological properties and intracellular calcium signaling in DRG neurons in the intact ganglion where the effects of somatic spike invasion can be studied as well.

Fundamentals of neurogastroenterology: basic science.

The focus of neurogastroenterology in Rome II was the enteric nervous system (ENS). To avoid duplication with Rome II, only advances in ENS neurobiology after Rome II are reviewed together with stronger emphasis on interactions of the brain, spinal cord, and the gut in terms of relevance for abdominal pain and disordered gastrointestinal function. A committee with expertise in selective aspects of neurogastroenterology was invited to evaluate the literature and provide a consensus overview of the Fundamentals of Neurogastroenterology textbook as they relate to functional gastrointestinal disorders (FGIDs). This review is an abbreviated version of a fuller account that appears in the forthcoming book, Rome III. This report reviews current basic science understanding of visceral sensation and its modulation by inflammation and stress and advances in the neurophysiology of the ENS. Many of the concepts are derived from animal studies in which the physiologic mechanisms underlying visceral sensitivity and neural control of motility, secretion, and blood flow are examined. Impact of inflammation and stress in experimental models relative to FGIDs is reviewed as is human brain imaging, which provides a means for translating basic science to understanding FGID symptoms. Investigative evidence and emerging concepts implicate dysfunction in the nervous system as a significant factor underlying patient symptoms in FGIDs. Continued focus on neurogastroenterologic factors that underlie the development of symptoms will lead to mechanistic understanding that is expected to directly benefit the large contingent of patients and care-givers who deal with FGIDs.

Microglia: a newly discovered role in visceral hypersensitivity?

Given the growing body of evidence for a role of glia in pain modulation, it is plausible that the exaggerated visceral pain in chronic conditions might be regulated by glial activation. In this study, we have investigated a possible role for microglia in rats with chronic visceral hypersensitivity and previously documented altered neuronal function. Experiments were performed on adult male Sprague-Dawley rats pre-treated with neonatal colon irritation (CI) and on control rats. Effects of fractalkine (FKN, a chemokine involved in neuron-to-microglia signaling) and of minocycline (an inhibitor of microglia) on visceral sensitivity were examined. Visceral sensitivity was assessed by recording the electromyographic (EMG) responses to graded colorectal distension (CRD) in mildly sedated rats. Responses to CRD were recorded before and after injection of FKN, minocycline or vehicle. Somatic thermal hyperalgesia was measured by latency of paw withdrawal to radiant heat. The pattern and intensity of microglial distribution at L6-S2 in the spinal cord was also compared in rats with CI and controls by fluorescence microscopy using OX-42. Results show that: (1) FKN significantly facilitated EMG responses to noxious CRD by >52% in control rats. FKN also induced thermal hyperalgesia in control rats, consistent with previous reports; (2) minocycline significantly inhibited EMG responses to noxious CRD by >70% in rats with CI compared to controls 60 min after injection. The anti-nociceptive effect of minocycline lasted for 180 min in rats with CI, reaching peak values 60 min after injection. Our results show that FKN enhances visceral and somatic nociception, whereas minocycline inhibits visceral hypersensitivity in chronically sensitized rats, which indicates a role for microglia in visceral hypersensitivity.

Differential effects of glutamate receptor antagonists on dorsal horn neurons responding to colorectal distension in a neonatal colon irritation rat model.

AIM: To investigate and compare the effects of spinal D-(-)-2-amino-7-phosphonoheptanoic acid (AP-7) and 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX), two glutamate receptor antagonists, on the responses of dorsal horn neurons to colorectal distension (CRD) in adult rats exposed to neonatal colon irritation (CI). METHODS: Hypersensitive SD rats were generated by CI during postnatal days 8, 10 and 12. Experiments on adult rats were performed using extracellular single-unit recording. The effects of spinal application of AP-7 (0.001, 0.01, 0.1, 1 mmoL) were tested on the CRD-evoked neuronal responses in 16 controls and 17 CI rats. The effects of CNQX (0.2, 2, 5, 10 micromoL) were also tested on the CRD-evoked responses of 17 controls and 18 CI neurons. RESULTS: (1) The average responses of lumbosacral neurons to all intensities of CRD in CI rats were significantly higher than those in control rats; (2) In control rats, AP-7 (0.01 mmoL) had no significant effect on the neuronal response to all intensities of CRD (20, 40, 60, 80 mmHg); while AP-7 (0.1 mmoL) inhibited the neuronal response to 80-mmHg CRD. By contrast, in CI rats, AP-7 (0.01-1 mmoL) attenuated the CRD-evoked neuronal responses to all distention pressures in a dose-dependent manner; (3) In control rats, CNQX (2 micromoL) had no significantly effect on the neuronal response to all intensities of CRD; however, CNQX (5 micromoL) significantly attenuated the responses to CRD in the 40-80 mmHg range. By contrast, CNQX (2-10 micromoL) significantly decreased the neuronal responses in CI rats to non-noxious and noxious CRD in a dose-dependent manner. CONCLUSION: Our results suggest that spinal N-methyl-D-aspartate (NMDA) and non-NMDA receptors may contribute to the processing of central sensitivity in a neonatal CI rat model, but they may play different roles in it.

Thalamic modulation of visceral nociceptive processing in adult rats with neonatal colon irritation.

Visceral pain originates from visceral organs in response to a noxious stimulus which, if prolonged, may lead to chronic changes in the neural network mediating visceral nociception. For instance, colon inflammation enhances the responses of neurons in the thalamus to colorectal distension (CRD), whereas lesion in the dorsal column (DC) reverses this neuronal sensitization, suggesting that the thalamus and the DC play major roles in chronic visceral pain. In this study, we used adult rats sensitized with neonatal painful colon irritation to reveal the contribution of the thalamus and the DC to neuronal hyperexcitability in a model of chronic visceral pain. We recorded the responses of lumbosacral neurons to CRD in control rats and in rats with colon irritation following stimulation or inactivation of the thalamus, and after DC lesion. Our results show that, first, neuronal responses to CRD decreased following thalamic stimulation in control rats, whereas, in rats with colon irritation, responses either decreased or increased; second, DC lesion attenuated or enhanced these effects in the positively or in the negatively modulated group of neurons, respectively; third, lidocaine injection in the thalamus reduced the responses to CRD in some of the neurons recorded in rats with colon irritation, but had no effect on those in control rats. Therefore, it is reasonable to speculate that plasticity in rats with colon irritation that may underlie chronic pain is sustained by feedback loops ascending in the DC and engaging the thalamus.

Intradermal capsaicin inhibits lumbar dorsal horn neuronal responses to colorectal distention.

The purpose of this study was to examine the effect of cutaneous inflammation on the responses of viscerosomatic convergent dorsal horn neurons to graded colorectal distension (CRD) and cutaneous mechanical stimulation. Responses of single viscerosomatic neurons in the lumbar dorsal horn of the rat spinal cord to CRD and to cutaneous stimuli were recorded before and 50 min after cutaneous inflammation induced by intradermal injection of capsaicin in the receptive field (RF) or in the ipsilateral and contralateral forepaw. Capsaicin injection in the RF induced an increase in the spontaneous activity of dorsal horn neurons, an expansion in the size of their RF and facilitated their responses to cutaneous stimuli. An injection placed in the center of the RF attenuated the responses to noxious CRD. Capsaicin injection in the forepaw caused a significant decrease in the responses to CRD but not to cutaneous stimuli. These results indicate that the inhibitory effects, evoked by cutaneous inflammation, can modulate the responses of dorsal horn neurons to CRD, independent of its effect on the responses to cutaneous mechanical stimuli.

Long-term sensitization of primary afferents in adult rats exposed to neonatal colon pain.

We have previously shown that colon irritation (CI) in neonates results in chronic visceral hypersensitivity in adult rats, associated with central neuronal sensitization in the absence of identifiable peripheral pathology. The purpose of this study is to assess the relative contribution of peripheral mechanisms to chronic visceral hypersensitivity by examining the changes in responses of primary afferents at thoracolumbar (TL) and lumbosacral (LS) spinal segments to graded colorectal distension (CRD). Afferent discharges were recorded at the cut distal ends of spinal dorsal roots (DRs) in adult control and CI rats. We found that: (1). the average threshold of activation of LS afferents decreased significantly in CI rats; (2). the responses of TL and LS afferents to CRD in CI rats were significantly greater than those in control; (3). the spontaneous activity of LS afferents in CI rats was significantly stronger than that in control; (4). in CI rats, the average responses to graded CRD of LS DRs were significantly higher than those of TL DRs; and (5). the number of both LS DRs and TL DRs activated by CRD in CI rats was significantly larger than control. In summary, the results show that chronic visceral hypersensitivity is associated with peripheral sensitization, as well as central sensitization. TL visceral afferents projecting seem to be more involved in the processing of sensitized nociceptive input from the colon than acute nociceptive input. However, LS afferents seem to be equally important in both sensitized and acute pain states.

Noxious visceral inputs enhance cutaneous tactile response in rat thalamus.

The current study investigates whether visceral nociceptive inputs affect tactile processing in the thalamic ventroposterior lateral nucleus in anesthetized rats by means of extracellular single unit recordings. Twenty out of the 44 neurons had their response to tactile stimulation increased by preceding nociceptive colorectal distension (CRD), and this influence appear more potent than the opposite effect, tactile on CRD response. There was a dynamic change of tactile response along with CRD response and the background activities as a function of repeated colorectal stimulation. The abnormal neuronal discharge in response to tactile stimulation in the condition of visceral nociception could contribute to the development of referred pain and allodynia.

Effect of tactile inputs on thalamic responses to noxious colorectal distension in rat.

Recent discoveries of visceral nociceptive inputs sharing the classical tactile pathway in the dorsal-column medial lemniscus system have opened a new venue for the investigation of somatovisceral interactions. The current study was designed to determine whether somatic innocuous inputs modulate visceral nociceptive transmission at the thalamic level. The investigation was carried out by means of extracellular single-unit recordings in the ventroposterior lateral nucleus of the thalamus in rats anesthetized with pentobarbital. Noxious visceral stimulation was achieved by reproducible colorectal distension (CRD, 20-80 mmHg) with a balloon catheter. Tactile stimulation was delivered by means of a feedback-controlled mechanical stimulator. The response of the neurons to CRD was compared before and after the conditioning procedure by giving tactile stimulation either immediately before CRD or overlapping it. Twenty-five ventroposterior lateral (VPL) thalamic neurons were found among numerous tactile-only neurons to have convergent inputs from both tactile and visceral sources. Their responses to CRD were excitatory (19), inhibitory (4), or bimodal. When cutaneous tactile stimuli were delivered before CRD, the responses were reduced in 18 cases. The reduction, however, was usually short-lasting, immediately following tactile stimulation and could not be enhanced by a prolonged conditioning procedure. It was unlikely to be attributable to neuronal habituation as the inverted procedure, CRD stimulation before tactile, often produced the opposite effect, that is, an enhanced response to skin stimulation. Repeated CRD could bring about sensitization of the responses of thalamic neurons as manifested by increased spontaneous discharge, lowered response threshold, and increased response level. Under such circumstances, the original effect of tactile stimulation on CRD responses could be weakened. In conclusion, tactile stimulation may in most circumstances inhibit thalamic neuronal responses to visceral nociceptive input produced by CRD. However, the effect appears to be mild and short-lasting at the individual neuronal level in the VPL thalamus.