Q. Greba, L. Munro and L. Kokkinidis 2001. The Involvement of Ventral Tegmental Area Cholinergic Muscarinic Receptors in Classically Conditioned Fear Expression as Measured with Fear-Potentiated Startle. Brain Research, 870, 135-141.

Abstract. Accumulating evidence suggests that dopamine (DA) neurons in the ventral tegmental area (VTA) contribute to the complex amygdala-based neurocircuitry that mediates fear-motivated behaviors. Because of acetylcholine's (ACh) role in DA neuronal activation, the involvement of VTA cholinergic muscarinic receptors in Pavlovian conditioned fear responding was evaluated in the present study. Fear-potentiated startle was used to assess the effects of intraVTA infused methylscopolamine on conditioned fear performance in laboratory rats. Application of this nonspecific muscarinic receptor antagonist to VTA neurons was observed to inhibit the ability of a conditioned stimulus (CS) previously paired with footshock to enhance the amplitude of the acoustic startle reflex. Doses of methylscopolamine that blocked conditioned fear expression did not alter baseline sensorimotor responding. These results identify ACh neurotransmission in the VTA as a potential excitatory mechanism underlying the fear-arousing properties of threatening environmental stimuli.



Q. Greba, and L. Kokkinidis, 2000. Peripheral and Intramygdaloid Administration of the Dopamine D1 Recpetor Anatagonist SCH 23390 Blocks Fear-Potentiated Startle but not Shock reactivity or the Shock Sensitization of Acoustic Startle. Behavioral Neuroscience, 114, 262-272.

Abstract. Central dopamine (DA) activity is thought to play a role in fear motivation. The aim of the present study was to assess the involvement of DA D1 receptors in emotional learning. The authors report that peripheral and intraamygdalar administration of the specific D1 receptor antagonist SCH 23390 blocked the acquisition of fear-potentiated startle. Analysis of shock reactivity during footshock administration revealed that the learning impairment could not be explained by a diminution in the aversive properties of the unconditioned stimulus. Additionally, systemic and intraamygdalar injection of SCH 23390 did not alter fear expression as measured with the shock sensitization of acoustic startle. The potential contribution of mesoamygdaloid DA to the acquisition and retrieval of conditioned fear responses is discussed.



D.L. Gelowitz and L. Kokkinidis, 1999. Enhanced Amygdala Kindling After Electrical Stimulation of the Ventral Tegmental Area: Implications for Fear and Anxiety. Journal of Neuroscience, 19, 1-5.

Abstract. Electrical kindling refers to the seizure-generating properties of brain stimulation. In addition to producing epilepsy, the reorganization of forebrain neurocircuitry associated with kindling contributes to psychiatric disturbances involving fear and anxiety. The amygdala is a limbic structure that kindles readily and regulates the complex neurocircuitry underlying emotional responding. Dopamine-containing ventral tegmental area (VTA) neurons, known to be activated by threatening environmental stimuli, are an important component of the amygdala-based fear network. Using amygdala kindling as an indicator of sensitization development, we report here that repeated low-current, high-frequency stimulation of the VTA provoked afterdischarge in the central amygdala and enhanced kindling rate. By establishing a fundamental link between VTA activation and neural excitability in the central amygdala, the present results are consistent with the possibility of a common process underlying epileptogenisis and the fear motivational consequences of amygdala and VTA kindling. Considering the established role of the VTA and the amygdala in emotional responding, such a sensitization mechanism might mediate exaggerated fearfulness.



Reynolds, J. N. J., B. I. Hyland, et al. (2001). A cellular mechanism of reward-related learning. Nature 413(6851): 67-70.

Abstract. Positive reinforcement helps to control the acquisition of learned behaviours. Here we report a cellular mechanism in the brain that may underlie the behavioural effects of positive reinforcement. We used intracranial self-stimulation (ICSS) as a model of reinforcement learning, in which each rat learns to press a lever that applies reinforcing electrical stimulation to its own substantia nigra. The outputs from neurons of the substantia nigra terminate on neurons in the striatum in close proximity to inputs from the cerebral cortex on the same striatal neurons. We measured the effect of substantia nigra stimulation on these inputs from the cortex to striatal neurons and also on how quickly the rats learned to press the lever. We found that stimulation of the substantia nigra (with the optimal parameters for lever-pressing behaviour) induced potentiation of synapses between the cortex and the striatum, which required activation of dopamine receptors. The degree of potentiation within ten minutes of the ICSS trains was correlated with the time taken by the rats to learn ICSS behaviour. We propose that stimulation of the substantia nigra when the lever is pressed induces a similar potentiation of cortical inputs to the striatum, positively reinforcing the learning of the behaviour by the rats.

Reynolds, J. N. J. and J. R. Wickens (2000). Substantia nigra dopamine regulates synaptic plasticity and membrane potential fluctuations in the rat neostriatum, in vivo. Neuroscience 99(2): 199-203.

Abstract. The spiny projection neurons of the neostriatum are a site at which dopamine inputs from the substantia nigra converge with excitatory inputs from the cerebral cortex. These two systems interact in certain learning and motor control mechanisms of the brain. We investigated these interactions using intracellular recording from spiny striatal neurons in urethane-anaesthetized rats. We found that acute dopamine depletion was associated with long-term depression of corticostriatal synaptic input. Electrical stimulation of the cortex which mimicked synchronous cortical input to striatal neurons also induced long-term depression of corticostriatal inputs. In intact control animals, but not in dopamine-depleted animals, this depression was prevented or reversed by concomitant stimulation of the substantia nigra. In agreement with previous in vitro studies, our in vivo findings show that long-term depression occurs in the corticostriatal pathway, and in addition show that it is regulated by dopaminergic inputs from the substantia nigra. This form of synaptic plasticity may therefore be important for understanding disturbances of the motor system seen in humans with Parkinson's disease.

L.F.B. Nicholson, R.L.M. Faull, H.J. Waldvogel and M. Dragunow. The regional, cellular and subcellular localization of GABAA/benzodiazepine receptors in the substantia nigra of the rat. Neuroscience, 1992, 50, 355-370

Abstract. The regional, cellular and subcellular distribution of GABAA/benzodiazepine receptors was investigated by light and electron microscopy in the rat substantia nigra. The regional distribution and density of GABAA/benzodiazepine receptor subtypes (Type I and II) was studied using quantitative receptor autoradiography following in vitro labelling of cryostat sections with tritiated ligands. This was followed by a detailed study of the cellular and subcellular distribution and localization of GABAA/benzodiazepine receptors by light and electron microscopy using immunohistochemical techniques with a monoclonal antibody (bd-17) to the beta 2,3 subunits of the GABAA/benzodiazepine receptor complex. Finally, in situ hybridization histochemistry using 35S-labelled oligonucleotide probes was used to demonstrate the cellular distribution of mRNA for the alpha 1 and alpha 2 GABAA receptor subunits in the substantia nigra. The results of the autoradiographic and immunohistochemical studies showed a close correspondence in the regional distribution of GABAA/benzodiazepine receptors in the substantia nigra. A moderate-to-high density of receptors was present throughout the full extent of the substantia nigra pars reticulata with a very low density of receptors in the substantia nigra pars compacta. Quantitative autoradiographic studies showed that: (i) the pars reticulata contained mainly central Type I receptors; (ii) the highest density of receptors was present in the caudal pars reticulata (200 +/- 38 fmol/mg) with successively lower densities of receptors in the middle (176 +/- 31 fmol/mg) and rostral (150 +/- 26 fmol/mg) levels of the pars reticulata; and (iii) the density of receptors in the pars reticulata was reduced by 34% following 6-hydroxydopamine-induced degeneration of dopaminergic pars compacta neurons. At the cellular level, GABAA/benzodiazepine receptor immunoreactivity was localized in a punctate fashion on dendrites and neuronal cell bodies in the pars reticulata. At the subcellular level, GABAA/benzodiazepine receptor immunoreactivity was associated with the pre- and postsynaptic membranes of axodendritic synaptic complexes along the length of small-to-large sized smooth dendrites in the pars reticulata. Two types of immunoreactive axodendritic synaptic complexes were identified: most (about 80%) immunopositive synapses showed equal staining of the pre- and postsynaptic membranes and were associated with small (less than 1.0 micron) axon terminals containing few mitochondria and small, round-to-pleomorphic vesicles in synaptic contact with small, peripheral dendrites; less frequently (about 20%) immunopositive synapses showed a marked immunoreactive thickening of the postsynaptic membrane and were associated with large (greater than 1.0 micron) axon terminals containing numerous mitochondria and mainly pleomorphic vesicles in synaptic contact with large mainstem dendrites

R.L.M. Faull, H.J. Waldvogel, L.F.B. Nicholson and B.J.L. Synek. The distribution of GABAA-benzodiazepine receptors in the basal ganglia in Huntington's disease and in the quinolinic acid lesioned rat. Prog. Brain Res., 99, 105-123, 1993 .

Abstract. The distribution of NADPH diaphorase staining in the human basal ganglia was compared in five cases who were neurologically normal with five cases who died with Huntington's disease. The normal cases showed an intense staining for NADPH diaphorase throughout all regions of the neuropil in the striatum (caudate nucleus, putamen and nucleus accumbens); the staining in the neuropil was largely homogeneous although a heterogeneous distribution was evident at rostral levels of the head of the caudate nucleus and in the nucleus accumbens where patches of reduced staining aligned with acetylcholinesterase-poor regions. The globus pallidus showed a moderately intense homogeneous pattern of staining for NADPH diaphorase. In comparison with control cases, sections of the striatum from the five cases with Huntington's disease showed a dramatic decrease in the intensity of NADPH diaphorase staining in the neuropil, especially in the caudate nucleus and putamen. In cases of early Huntington's disease where no discernible loss of neurons was seen [grade 0 using the grading criteria of Vonsatell et al. (1985) J. Neuropath. exp. Neurol. 44, 559-577], there was a marked heterogeneous pattern of staining in the caudate nucleus and putamen showing a patchy loss of NADPH diaphorase in the neuropil. This resulted in clearly delineated islands of greatly reduced staining surrounded by a matrix of moderately reduced staining; the patches of greatly reduced staining corresponded with acetylcholinesterase-poor striosomes. In cases of more advanced neuropathology (grades 1 and 2) the loss of NADPH diaphorase staining in the neuropil was even more marked, affecting both acetylcholinesterase-poor and acetylcholinesterase-rich regions of the caudate nucleus and putamen and resulting in an almost homogeneous loss of staining in these striatal regions. Despite this marked loss of staining in the neuropil, the numbers of NADPH diaphorase-stained neuronal somata in the striatum in Huntington's cases appeared comparable to those in the control cases. In the globus pallidus of one of the advanced Huntington's disease cases there appeared to be a minimal loss of NADPH diaphorase staining; however, staining in the other regions of the brain which were examined was similar to that in the control cases. These findings demonstrate a progressive compartmental loss of NADPH diaphorase in the neuropil of the human striatum in Huntington's disease which correlates with the extent of neurodegeneration; early in the disease the loss of neuropil staining is first evident in the striosome compartment, then followed by an additional loss in the matrix compartment in more advanced cases of the disease.

Louise F.B. Nicholson , John C. Montgomery and Richard L.M. Faull. GABA, muscarinic cholinergic, excitatory amino acid, neuortensin and opiate binding sites in the octavolateralis column and cerebellum of the skate Raja nasuta (Pices: Rajidae). Brain Res., 652, 40-48, 1994

Abstract. As part of a study of signal processing in the electro- and mechanosensory systems we have screened the octavolateralis column of the skate for GABAA, muscarinic cholinergic, excitatory amino acid, neurotensin and opiate binding sites using autoradiography following in vitro labelling of cryostat sections with tritiated ligands. The presence and distribution of these binding sites is compared between the octavolateralis column and the corpus cerebellum. GABAA binding sites were located in high concentrations in the granule cell regions of the cerebellum and octaval columns, with much lower concentrations in the Purkinje cell layer of the corpus cerebellum. Little or no labelling was evident in all molecular layer areas. Displacement studies using the discriminating ligand CL218,872 indicated that the GABAA binding sites were predominantly of the GABAA/benzodiazepine Type II variety. M1 muscarinic cholinergic binding sites were found in high concentrations in all granule cell areas and in lower concentrations in the molecular layer of the octavolateralis column, with an absence of labelling in the molecular layer of the corpus cerebellum. Kainic acid and AMPA binding sites were present in very high concentrations in all molecular layer areas. Glutamate binding was present in the molecular layer of the octavolateralis column and in some restricted regions of the dorsal granular ridge, whereas phencyclidine binding sites were sparse or absent. Neurotensin binding sites were strongly present in all granule cell areas and evident in the molecular layer of the octavolateralis column. There was evidence for opiate binding sites in the molecular layer of both the dorsal and medial octavolateralis nucleus.

L.F.B. Nicholson, R.L.M. Faull, H.J. Waldvogel and M. Dragunow. GABA and GABAA receptor changes in the substantia nigra of the rat following quinolinic acid lesions in the striatum closely resemble Huntington's disease. Neuroscience, 66, 507-521, 1995

Abstract. GABA and GABAA receptors have been studied in the substantia nigra of the rat following quinolinic acid lesions in the striatum. The regional distribution of GABA and GABAA receptors was investigated using immunohistochemical techniques with monoclonal antibodies to GABA and to the beta 2.3 subtypes of the GABAA receptor complex. The distribution, density and cellular localization of GABAA receptors were studied using quantitative receptor autoradiography and 6-hydroxydopamine-induced degeneration of dopaminergic pars compacta neurons. The subunit configuration of GABAA receptors was investigated using in situ hybridization histochemistry and subunit subtype-specific oligonucleotide probes. The results showed that in the normal substantia nigra GABA and GABAA receptors were mainly localized within the pars reticulata. GABAA receptors were mainly of the BZI variety, had a subunit subtype configuration that included alpha 1 and beta 2.3 subtypes, and showed a rostrocaudal gradient in the density of receptors; the density of receptors in the caudal third was 56% higher than that in the rostral third of the pars reticulata. Following quinolinic acid-induced degeneration of the striatonigral pathway, there was a marked loss of GABA immunoreactivity and a 59% increase in the density of GABAA receptors in the substantia nigra pars reticulata. There was a corresponding regional topography in the pattern of loss of GABA immunoreactivity and in the pattern of increase in GABAA receptors in the pars reticulata; the topography varied with the size and placement of the lesion in the striatum and correlated with the known topographical organization of the striatonigral projection. The quantitative autoradiographic results showed that following quinolinic acid lesions in the striatum: (i) the greatest increase in the density of GABAA receptors occurred in the middle third (91% increase) of the pars reticulata; (ii) the receptors were mainly of the GABAA/BZI variety; and (iii) 6-hydroxydopamine-induced degeneration of the dopaminergic pars compacta neurons did not significantly affect the density of receptors, indicating that the increased receptor binding was mainly localized on non-dopaminergic pars reticulata neurons. The immunohistochemical and in situ hybridization studies showed that, as in the normal substantia nigra, GABAA receptors in the substantia nigra pars reticulata on the lesioned side contained the alpha 1 and beta 2.3 GABAA receptor subtypes; the alpha 1 and beta 2.3 subtypes (but not the alpha 2) were increased after quinolinic acid lesions

R.L.M.Faull, H.J.Waldvogel, L.F.B. Nicholson, M.N.Williams and M.Dragunow. Huntington's disease and neural transplantation: GABAA receptor changes in the basal ganglia in Huntington's disease in the human brain and in the quinolinic acid lesioned rat model of the disease following fetal neuron transplants. In: Neurotransmitters in the Human Brain, Eds. D.Tracey, J.Stone and G.Paxinos. Plenum Press, pp. 173-197, 1995.

L.F.B. Nicholson and R.L.M. Faull. GABAA receptor subunit subtypes in the human putamen and globus pallidus in Huntington's disease - an in situ hybridization study. In The Basal Ganglia V. (eds Ohye C., Kimura M. and McKenzie J.), Plenum Press, New York, pp 433-440, 1996.

LFB Nicholson, HJ Waldvogel and RLM Faull. GABAA receptor subtype changes in the substantia nigra of the rat following quinolinic acid lesions in the striatum: a correlative in situ hybridization and immunohistochemical study. Neuroscience, 1996, 74, 89-98.

Abstract. This study investigates the pattern of distribution of GABAA receptor subunit subtypes in the substantia nigra of the rat using in situ hybridization techniques and immunohistochemistry at the light microscopic level following unilateral quinolinate lesions in the striatum. The main purpose of this study was to first identify the variety and regional distribution of GABAA receptor subtype messenger RNAs in the normal substantia nigra and, second, to determine if this pattern and level of expression of GABAA receptor subtypes in the substantia nigra is affected following quinolinate-induced degeneration of the GABAergic striatonigral projection neurons. The study is based on a comparison of adjacent sections using: (i) in situ hybridization and oligonucleotide probes selective for 13 of the GABAA receptor subunits; and (ii) immunohistochemistry and antibodies specific to three protein subunits of the GABAA receptor complex. The results show that the GABAA receptor in the normal substantia nigra pars reticulata has a molecular configuration comprising of the alpha 1, beta 2, and gamma 2 subtypes and that following quinolinate lesions of the striatum the subtype configuration of the GABAA receptors remains unaltered, but that there is a marked increase in the level of expression of the alpha 1, beta 2 and gamma 2 subtypes. In confirmation of these findings, the immunohistochemical results show increased immunoreactivity for the alpha 1, beta 2,3 and gamma 2 GABAA receptor subtypes in the substantia nigra following degeneration of GABAergic striatonigral neurons. The details of these findings are discussed with reference to previous studies and with regard to the implications that these results may have for specific GABAergic neurodegenerative diseases of the human basal ganglia, such as Huntington's disease.

RLM Faull, HJ Waldvogel, LFB Nicholson, MN Williams and M Dragunow. Huntington's disease and neural transplantation. In: The Basal Ganglia V. (eds Ohye C., Kimura M. and McKenzie J.), Plenum Press, New York, pp 445-454, 1996.

JC Vis, LFB Nicholson, RLM Faull, WH Evans, NJ Severs and CR Green. Gap junction distribution in Huntington's diseased brain indicates a role for connexin43 in spatial buffering to promote neuronal survival. Cell Biology International Special Edition - Gap Junctions in the Brain, 22, 837 - 847, 1998.

KL Brickell, LFB Nicholson, HJ Waldvogel and RLM Faull. Chemical and anatomical changes in the striatum and substantia nigra following quinolinic acid lesions in the striatum of the rat: a detailed time course of the cellular and GABAA receptor changes. Journal of Chemical Neuroanatomy, 17, 75 - 97, 1999.

Abstract. The pattern and time-course of cellular, neurochemical and receptor changes in the striatum and substantia nigra were investigated following unilateral quinolinic acid lesions of the striatum in rats. The results showed that in the central region of the striatal lesion there was a major loss of Nissl staining of the small to medium sized cells within 2 h and a substantial loss of neuronal staining within 24 h after lesioning. Immunohistochemical studies showed a total loss of calbindin immunoreactivity, a known marker of GABAergic striatal projection neurons, throughout the full extent of the quinolinic acid lesion within 24 h. Similarly, within 24 h, there was a total loss of somatostatin/neuropeptide Y cells in the centre of the lesion but in the periphery of the lesion these cells remained unaltered at all survival times. Striatal GABA(A) receptors remained unchanged in the lesion for 7 days, and then declined in density over the remainder of the time course. Glial fibrillary acidic protein immunoreactive astrocytes were present in the periphery of the lesion at 7 days, occupied the full extent of the lesion by 4 weeks, and remained elevated for up to 2 months. In the substantia nigra, following placement of a striatal quinolinic acid lesion, there was: a loss of substance P immunoreactivity within 24 h; a marked astrocytosis evident from 1-4 weeks postlesion; and, a major increase in GABA(A) receptors in the substantia nigra which occurred within 2 h postlesion and was sustained for the remainder of the time course (15 months). This study shows that following quinolinic acid lesions of the striatum there is a major loss of calbindin and somatostatin/neuropeptide Y immunoreactive cells in the striatum within 24 h, and a marked increase in GABA(A) receptors in the substantia nigra within 2 h. These findings are similar to the changes in the basal ganglia in Huntington's disease and provide further evidence supporting the use of the quinolinic acid lesioned rat as an animal model of Huntington's disease.

P. Laslo, J. Lipski, LFB Nicholson, GB Miles, and GD Funk. Calcium binding proteins in motoneurons at low and high risk for degeneration in ALS. NeuroReport, 11 (15),3305-3308, 2000.

Abstract. Recent reports challenge the hypothesis that expression of calcium binding proteins contributes to the greater resistance of some motoneurons to degeneration in amyotrophic lateral sclerosis (ALS). We therefore re-examined, using immunohistochemistry, the expression of calbindin, calretinin and parvalbumin in vulnerable (hypoglossal, XII; and cervical spinal) and resistant (oculomotor, III) motoneurons of adult rats. Calbindin immunoreactivity was lacking in motor nuclei but strong in the dorsal horn. Calretinin was expressed in spinal, but not III or XII, motoneurons. Parvalbumin immunoreactivity, tested with a polyclonal antibody, was intense in spinal and III, but not XII, motoneurons; however, no staining in the ventral horn was observed with a monoclonal antibody. Differential expression of calretinin and parvalbumin within vulnerable motoneurons suggests that immunoreactivity for these proteins is not a reliable marker for resistance to degeneration in ALS.

P. Laslo, J. Lipski, LFB Nicholson, GB Miles, and GD Funk. GluR2 AMPA receptor subunit expression in motoneurons at low and high risk for degeneration in Amyotrophic lateral sclerosis. Experimental Neurology, 169, 461-471, 2001.

Abstract. Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder that results in selective degeneration of most, but not all, groups of motoneurons. The greater susceptibility of vulnerable motoneurons to glutamate excitotoxicity and neurodegeneration has been hypothesized to result from their lower expression of the GluR2 AMPA receptor subunit under control conditions, which renders these receptors permeable to calcium. To address the question of whether there is differential expression of the GluR2 subunit in motoneurons, we compared in normal adult rats expression of GluR2 mRNA and protein within two cranial motor nuclei that are either resistant (III; oculomotor nucleus) or vulnerable (XII; hypoglossal nucleus) to degeneration in ALS. RT-PCR analysis of tissue punched from III and XII motor nuclei detected mRNA for all AMPA subunits (GluR1-R4). In situ hybridization demonstrated no significant difference in GluR2 mRNA expression between III and XII nuclei. Immunohistochemical examination of GluR2 (and GluR4) protein levels demonstrated a similar pattern of the subunit expression in both motor nuclei. This equivalent expression of GluR2 mRNA and protein in motoneurons that differ in their vulnerability to degeneration in ALS suggests that reduced expression of GluR2 is not a factor predisposing motoneurons to degeneration.

C.G. Fonseca. C.R.Green and L.F.B.Nicholson. Upregulation in astrocytic connexin 43 gap junction levels may exacerbate generalized seizures in mesial temporal lobe epilepsy. Brain Research, 929, 105 - 116, 2002

Abstract. Following brain injury, and during the process of neurodegeneration, a reactive astrocytic proliferation occurs. This is accompanied by an increase in the synthesis of neuropeptides, cytokines, growth factors and glial fibrillary acidic protein (GFAP), a cell-specific marker for reactive astrocytes. Astrocytes are extensively coupled by gap junctions of the Cx43 connexin subtype. Several studies have shown that in severe trauma, coupling between astrocytes may add to the spread of the damaged area. In this study we ask whether the astrocytosis which is a feature of other neurodegenerative diseases also occurs in mesial temporal lobe epilepsy (MTLE) and whether it is accompanied by an increase in astrocytic communication through an upregulation of Cx43 gap junction channel proteins. In order to examine the astrocytic response and the expression pattern of Cx43 protein, double immunohistochemical labeling studies were undertaken using antibodies against GFAP and Cx43 applied to human hippocampal tissue resected from patients with MTLE, and to normal human control hippocampal tissue. Immunofluorescent labeling of astrocytes and Cx43 was examined using confocal laser scanning microscopy. The images obtained were quantitatively analysed and reconstructed using three-dimensional volume rendering. The results of this study have established that not only is astrocytosis greater in MTLE-affected tissues than previously suggested, but it is accompanied by a highly significant increase in astrocytic Cx43 protein levels. We hypothesize that this surprisingly large upregulation in Cx43 may exacerbate generalized seizures in the progression of MTLE.

L. Ma, R. Carter, A. J.Morton and L. F. B.Nicholson. RAGE is upregulated in rat hippocampus following hypoxic-ischemic brain injury. In Press - Brain Research

L. F. B. Nicholson. Processing Human Brain Tissue for in situ hybridization with radiolabel oligonucleutides, 103-113. In In situ hybridization Protocols for the brain. eds. W.Wisden and B.J.Morris, Academic Press, 2002

The Basal Ganglia VII. (eds Nicholson, Louise FB and Faull, RLM), Advances in Behavioral Biology, Vol. 52. Kluwer Academic/Plenum Press, New York pp 638, 2002..

Ping Liu, Yiwen Zheng, Jaimee King, Cynthia L. Darlington, Paul F. Smith. Nitric oxide synthase and arginase expression in the vestibular nucleus and hippocampus following unilateral vestibular deafferentation in the rat. Brain Res. 2003 Mar 14;966(1):19-25.

Abstract. The aim of this study was to investigate the possible relationship between changes in neuronal and endothelial nitric oxide synthase (nNOS and eNOS) and arginase expression in the vestibular nucleus complex and the hippocampus (CA1, CA2/3 and the dentate gyrus (DG) at 10 h or 2 weeks following a unilateral vestibular deafferentation (UVD) in rats. There were no significant differences in nNOS or arginase II expression in the ipsilateral or contralateral VNC at either 10 h or 2 weeks post-UVD. For eNOS, there was only a significant decrease in expression in the ipsilateral VNC at 2 weeks post-UVD (P,0.01). In the hippocampus, the only significant difference in nNOS expression was a decrease in the ipsilateral DG at 2 weeks post-UVD (P,0.05). There was a significant decrease in eNOS expression in the contralateral CA2/3 region at 10 h post-UVD (P,0.01). The only other significant change in eNOS was an increase in the contralateral DG at 10 h post-UVD (P,0.01). Although arginase II was expressed in all regions of the hippocampus, there were no significant differences in arginase II expression at any time point following UVD. These results suggest that the changes in NOS expression that occur in the VNC and hippocampus following UVD are not correlated with one another or with changes in arginase II.

Catherine M. Gliddon, Cynthia L. Darlington, Paul F. Smith. Activation of the hypothalamic-pituitary-adrenal axis following vestibular deafferentation in pigmented guinea pig. Brain Research 964 (2003) 306-310

Abstract. Twelve male pigmented guinea pigs underwent either a unilateral vestibular deafferentation (UVD) (n56) or sham operation (n56). Compared to the pre-operated salivary cortisol concentrations, the UVD operation resulted in a significant increase in night cortisol concentrations (P,0.05) and a significant interaction between the night cortisol concentration and time (P,0.05). There was no significant difference between the pre- and post-UVD morning salivary cortisol concentrations; nor between the pre- and post-sham morning or night salivary cortisol concentrations. This study suggests that the ocular-motor and postural syndrome is causing the activation of the hypothalamic-pituitary-adrenal (HPA) axis.

Ping Liu, Yiwen Zheng, Paul F. Smith, and David K. Bilkey. Changes in NOS Protein Expression and Activity in the Rat Hippocampus, Entorhinal and Postrhinal Cortices After Unilateral Electrolytic Perirhinal Cortex Lesions. HIPPOCAMPUS 13:561-571 (2003).

Abstract. The integrity of the perirhinal cortex is critical for certain types of learning and memory. One important issue relating to the function of this region is its interaction with other brain areas that play a role in memory processing. This study investigates the time course of changes in activity and protein expression of nitric oxide synthase (NOS), which transforms L-arginine into nitric oxide (NO) and citrulline, in the hippocampus and the entorhinal and postrhinal cortices after unilateral electrolytic lesions of the perirhinal cortex. Electrolytic lesions of the perirhinal cortex resulted in long lasting changes in NOS activity and protein expression in the entorhinal and postrhinal cortices (<2 weeks post-lesion). In contrast, there was a small and transient decrease in nNOS expression (with no change in NOS activity) in the dorsal portion of the hippocampus. iNOS was not expressed in any region examined at any time point. These .ndings provide the .rst evidence that electrolytic lesions of the perirhinal cortex can result in long-term neurochemical changes in its anatomically related structures. Given that NO has been implicated in neuroplasticity processes, the interpretation of memory impairments induced by electrolytic lesions of the perirhinal cortex (and possibly, therefore, other brain regions) need to be considered with regard to these findings.

Karyn M. Maclennan, Yiwen Zheng, Philip W. Sheard, Sheila M. Williams, Cynthia L. Darlington, and Paul F. Smith. Adrenalectomy-Induced Cell Death in the Dentate Gyrus: Further Characterisation Using TUNEL and Effects of the Ginkgo biloba Extract, EGb 761, and Ginkgolide B. HIPPOCAMPUS 13:212-225 (2003).

Abstract. This study investigated the potential neuroprotective effects of the Ginkgo biloba extract, EGb-761, and ginkgolide B, on adrenalectomy (ADX)-induced cell death in the dentate gyrus (DG). Adrenalectomised, sham surgery-treated, and naive controls received either EGb- 761 (25, 50, or 100 mg/kg), 0.9% saline vehicle control, ginkgolide B (10 or 25 mg/kg), or a polyethylene glycol vehicle control, i.p, daily for 6 days postsurgery. Cell death in the DG was determined by in situ labelling of DNA fragments, using the TUNEL method; sections were counterstained with hematoxylin. Radioimmunoassay was used to con.rm a decrease in plasma corticosterone (CORT) after ADX. TUNEL-positive granule cells were observed in the DG at 1 week, but not at 24 h, post-ADX. The rate of granule cell death at this time was highest in the suprapyramidal blade and increased in a crest tip and a rostrotemporal gradient. Whereas CORT replacement completely prevented the occurrence of TUNEL-positive granule cells, EGb-761 and ginkgolide B did not, at any of the doses used. These results suggest that these drugs may not have substantial neuroprotective effects in the ADX model of neurodegeneration.

Arata Horii, Paul F. Smith, Cynthia L. Darlington. Application of real-time quantitative polymerase chain reaction to quantification of glutamate receptor gene expression in the vestibular brainstem and cerebellum. Brain Research Protocols 9 (2002) 77-83.

Abstract. Reverse transcription-polymerase chain reaction (RT-PCR) is a powerful tool to detect specific gene expression from a small amount of tissue, which is superior to the traditional RNA assays such as Northern blotting and in situ hybridization (ISH) in terms of sensitivity. However, conventional RT-PCR is not suitable for quantification due to its exponential nature. Recently, a real-time quantitative PCR method has been developed to overcome the weak points of RT-PCR, e.g. quantification. Here we describe the use of real-time quantitative PCR using a fluorescent TaqMan probe, to study the regional differences in expression of glutamate receptor subunit / subtype genes (NR1, NR2A, GluR2, KA2, mGluR1, mGluR7) in the central vestibular system including the vestibular nucleus complex, inferior olive and cerebellar flocculus.We found that real-time quantitative PCR yielded similar results to other techniques such as ISH but offered several advantages in terms of relative speed and ability to detect low levels of gene expression. We suggest that real-time quantitative PCR is a useful method to study gene expression for other neurotransmitter receptors in the vestibular brainstem and cerebellum, and is also expected to be more accurate to assess the changes in gene expression following any treatment.

P. Liu, Y. Zheng, J. King, C. L. Darlington And P. F. Smith. Long-Term Changes In Hippocampal N-Methyl-D-Aspartate Receptor Subunits Following Unilateral Vestibular Damage In Rat. Neuroscience 117 (2003) 965-970.

Abstract. Previous studies have indicated that damage to the peripheral vestibular system results in dysfunction of hippocampal place cells and an impairment of spatial learning and memory. The aim of this study was to determine whether lesions of one vestibular labyrinth (unilateral vestibular deafferentation, UVD) result in changes in the expression of the NR1 and NR2A subunits of the N-methyl-D-aspartate (NMDA) receptor, and the GluR2 subunit of the _-amino-3- hydroxy-5-methyl-4-isoxazole propionate receptor, in subregions of the rat hippocampus (CA1, CA2/3 and the dentate gyrus) at 10 h or 2 weeks following UVD. Compared with sham surgery controls and anaesthetic controls, the expression of the NR1 subunit was significantly reduced in the ipsilateral CA2/3 region at 2 weeks post-UVD. The expression of the NR2A subunit was also significantly reduced in the ipsilateral CA2/3 and, to a smaller extent, in the contralateral CA2/3 region, at 2 weeks post-UVD. The only other change in NR2A expression was an increase in the ipsilateral CA1 at 10 h post-UVD. No other changes in NR1, NR2A or GluR2 expression were observed in any hippocampal subregion, at any time point, or in cortical tissue at any time point. These results suggest that UVD may result in long-term changes in NMDA receptor subunit expression in the rat hippocampus.

Arata Horii, Paul F. Smith, Cynthia L. Darlington. Quantitative changes in gene expression of glutamate receptor subunits/subtypes in the vestibular nucleus, inferior olive and flocculus before and following unilateral labyrinthectomy in the rat: real-time quantitative PCR method. Exp Brain Res (2001) 139:188-200.

Abstract. Spontaneous recovery from the oculomotor and postural symptoms of unilateral labyrinthectomy (UL) is known as vestibular compensation, which is a useful model for investigation of the mechanisms of lesion- induced CNS plasticity. In the present study, to elucidate the molecular biological basis of vestibular compensation, we investigated changes in the mRNA expression of glutamate receptor subunit/subtypes in the rat central vestibular system, including the vestibular nucleus complex (VNC), inferior olive (IO), and cerebellar flocculus following UL, using a real-time quantitative polymerase chain reaction (PCR) method. In normal control animals, regional differences in the expression of several glutamate receptor subunit/subtypes, e.g., NR1 and NR2A subunits of the N-methyl-D-aspartic acid (NMDA) receptor, GluR2 and KA2 subtypes of non-NMDA receptors, and mGluR1 and mGluR7 metabotropic glutamate receptors, were consistent with previous results from studies using in situ hybridization histochemistry, suggesting that the real-time quantitative PCR method was a reliable procedure for evaluation of changes in mRNA expression. In the vestibular nucleus complex, NR2A, GluR2 and mGluR7 mRNA were ipsilaterally downregulated by 6 h following UL (P<0.05, P<0.05 and P<0.01, respectively). In the inferior olive, no changes in gene expression were observed. In the ipsilateral flocculus, KA2 mRNA expression was increased by 50 h post-UL (P<0.05). However, in the contralateral flocculus, mGluR1 mRNA was downregulated by 6 h post-UL (P<0.005). Both the increase in KA2 mRNA expression in the ipsilateral flocculus and the decrease in mGluR1 mRNA expression in the contralateral flocculus may have had the effect of reducing Purkinje cell inhibition of ipsilateral VNC neurons, thereby contributing to the rebalancing of spontaneous resting activity between the ipsilateral and contralateral VNCs. It is suggested that such changes in the activities of the floccular-VNC pathways may be important to the vestibular compensation process.

Paul F. Smith, Yiwen Zheng, Sylvia Paterson And Cynthia L. Darlington. The Contribution of Nitric Oxide to Vestibular Compensation: Are There Species Differences? Acta Otolaryngol 2001; Suppl 545: 57-60.

Abstract. Nitric oxide (NO) has been implicated in the processes by which animals recover from peripheral vestibular damage (''vestibular compensation''). However, there is little systematic data available on the effects of NO inhibition on the vestibular compensation process. In the present study we administered the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) using a subcutaneous osmotic minipump and examined its effects on the compensation of spontaneous nystagmus (SN), yaw head tilt (YHT) and roll head tilt (RHT) in guinea pigs. Following unilateral labyrinthectomy (UL), treatment with 5, 10, 50 or 100 mM L-NAME had no effect on the expression of any of these symptoms or their rate of compensation. By contrast, pre-UL treatment with 100 mM L-NAME resulted in a decrease in SN frequency at 10 h post-UL and an increase in its rate of compensation. Lower concentrations had no effect on SN. Pre-UL treatment with L-NAME had no signi. cant effect on YHT or RHT at any particular time point. Analysis of NOS activity demonstrated that the highest concentration of L-NAME inhibited NOS activity in the contralateral vestibular nucleus complex, bilateral cerebellum and bilateral cortices. These results suggest that L-NAME may have different effects on vestibular compensation in guinea pigs compared to other species, such as the rat and frog.

Y. Zheng, A. Horii,. Appleton, C. L. Darlington And P. F. Smith. Damage To The Vestibular Inner Ear Causes Long-Term Changes In Neuronal Nitric Oxide Synthase Expression In The Rat Hippocampus. Neuroscience Vol. 105, No. 1, pp. 1-5, 2001.

Abstract. The vestibular inner ear detects head acceleration and initiates compensatory eye movement and postural re£exes that help keep the visual image of the world stable on the retina, and maintain balance, during unexpected head movement. The most primitive vestibular systems are estimated to have evolved more than 500 million years ago and in mammalian and submammalian species the vestibular re£exes are mediated by basic brainstem pathways (see Wilson and Melvill Jones, 1979 for review). Although the contributions of the vestibular system to higher cognitive function have generally received less attention than its re£exive roles, vestibular sensory information is transmitted to higher centres in the brain and humans with vestibular damage are known to experience debilitating perceptual illusions (see Curthoys and Halmagyi, 1995; Berthoz, 1996 for reviews). Increasing behavioural and neurophysiological evidence suggests that the hippocampus uses information from the vestibular inner ear in order to build up maps of space that can be used in the development of spatial memory during learning tasks (McNaughton et al., 1991; Chapuis et al., 1992; Wiener and Berthoz, 1993; O'Mara et al., 1994; Wiener et al., 1995; Gavrilov et al., 1995; Stackman and Taube, 1996; Vitte et al., 1996; Taube et al., 1996; Save et al., 1998; Peruch et al., 1999; Cuthbert et al., 2000; Russell et al., 2000). However, to date, there has been no indication of the long-term neurochemical e°ects of the loss of vestibular input on hippocampal function. Since nitric oxide has been implicated in the mechanisms of hippocampal synaptic plasticity associated with the development of short-term memory (e.g. Schuman and Madison, 1991; Schuman et al., 1994; Arancio et al., 1996; Wu et al., 1997; Lu et al., 1999), we examined whether changes occur in the activity and expression of the enzymes responsible for nitric oxide production (nitric oxide synthases) in subregions of the rat hippocampus at di°erent times following unilateral peripheral vestibular lesions, using western blotting and radioenzymatic assays. We found a decreased expression of neuronal nitric oxide synthase in the ipsilateral dentate gyrus at 2 weeks following the vestibular damage and not before, that may be related to the long-term e°ects of the loss of vestibular input on hippocampal function. These results support the hypothesis that head movement and position information derived from the vestibular inner ear may be important for the normal function of the hippocampus

Ping Liu , David K. Bilkey , Cynthia L. Darlington , Paul F. Smith. Cannabinoid CB1 receptor protein expression in the rat hippocampus and entorhinal, perirhinal, postrhinal and temporal cortices: regional variations and age-related changes. Brain Research 979 (2003) 235–239.

Abstract. Cannabinoids have been shown to disrupt memory processes and these effects occur primarily through cannabinoid CB1 receptors in the brain. The present study investigates, for the first time, the regional variations and age-related changes in CB1 protein expression in the hippocampus and its neighbouring entorhinal, perirhinal, postrhinal and temporal cortices using Western blotting. In young adult rats, CB1 protein was highly expressed in the hippocampus and within the hippocampus, the greatest density of CB1 protein was located in CA1. When a comparison was made between young (4-month-old) and aged (24-month-old) rats, CB1 protein expression was significantly increased in the aged entorhinal and temporal cortices and was significantly decreased in the aged postrhinal cortex. The present study demonstrates region-specific changes in CB1 protein expression during ageing and further suggests that cannabinoid CB1 receptors may contribute to the aging process.

P. LIU, P. F. SMITH, I. APPLETON,C. L. DARLINGTON, AND D. K. BILKEY. Regional Variations And Age-Related Changes In Nitric Oxide Synthase And Arginase In The Sub-Regions Of The Hippocampus. Neuroscience 119 (2003) 679–687.

Abstract. L-arginine can be metabolised by nitric oxide synthase (NOS) with the formation of L-citrulline and nitric oxide (NO), or arginase with the production of L-ornithine and urea. In contrast to studies showing a potential involvement of NOS/NO in the aging process, the role of arginase has not been well documented. The present study investigates for the first time the regional variations and age-related changes in both NOS and arginase in sub-regions of the hippocampus. In young adult rats, although the total NOS activity was not significantly different across the hippocampal CA1, CA2/3 and the dentate gyrus (DG) sub-regions, the total arginase activity showed a clear regional variation with the highest level in DG. Western blotting revealed that the highest levels of neuronal NOS (nNOS) and endothelial NOS (eNOS) proteins were located in CA1. Arginase I is expressed at a very low level in the brain (the whole hippocampus) as compared with the liver. By contrast, arginase II protein shows an extremely high expression in the brain with little or no expression in the liver. There was no regional variation in arginase I or arginase II protein expression across the sub-regions of the hippocampus. When a comparison was made between young (4-month-old) and aged (24-month-old) rats, a significant increase in total NOS activity was found in DG and significant decreases in arginase activity were observed in the CA1 and CA2/3 regions in the aged animals. Western blotting further revealed a dramatic decrease in eNOS protein expression in aged CA2/3 with no age-associated changes in nNOS, arginase I and II protein expression in any region examined. Interestingly, evidence of activity or protein expression of the inducible isoform of NOS (iNOS) was not detected in any tissue from either group. The present results, in conjunction with previous findings, support the contribution of NOS/NO to aging but question the involvement of iNOS in the normal aging process. Region-specific changes in arginase suggest that this enzyme may also contribute to aging.

Noah A. Russell, Arata Horii, Paul F. Smith, Cynthia L. Darlington, and David K. Bilkey. Long-Term Effects of Permanent Vestibular Lesions on Hippocampal Spatial Firing. The Journal of Neuroscience, July 23, 2003 • 23(16):6490–6498.

Abstract. The hippocampus is thought to be important for spatial representation processes that depend on the integration of both self-movement and allocentric cues. The vestibular system is a particularly important source of self-movement information that may contribute to this spatial representation. To test the hypothesis that the vestibular system provides self-movement information to the hippocampus, rats were given either a bilateral labyrinthectomy (n.6) or a sham surgery (n.6), and at least 60 d after surgery hippocampal CA1 neurons were recorded extracellularly while the animals foraged freely in an open arena. Recorded cells were classified as complex spiking (n. 80) or noncomplex spiking (n.33) neurons, and their spatial firing fields (place fields) were examined. The most striking effect of the lesion was that it appeared to completely abolish location-related firing. The results of this and previous studies provide converging evidence demonstrating that vestibular information is processed by the hippocampus. The disruption of the vestibular input to the hippocampus may interfere with the reconciliation of internal self-movement signals with the changes to the external sensory inputs that occur as a result of that movement. This would disrupt the ability of the animal to integrate allocentric and egocentric information into a coherent representation of space.

Yiwen Zheng, Arata Horii, Paul F. Smith, Cynthia L. Darlington. Differences in NOS protein expression and activity in the rat vestibular nucleus following unilateral labyrinthectomy. Molecular Brain Research 88 (2001) 166-170.

Abstract. We used Western blotting to analyse the expression of different isoforms of nitric oxide synthase (NOS) in the rat vestibular nucleus complex (VNC) at various times following unilateral vestibular deafferentation (UVD), together with a radioenzymatic assay to compare NOS activity at the same time points. nNOS expression did not change significantly in the ipsilateral or contralateral VNC at any time following UVD. However, eNOS expression decreased significantly (P<0.05) in the contralateral VNC at 6 h post-UVD, recovering to normal levels by 50 h. iNOS was not expressed at any time following UVD. NOS activity demonstrated a significant increase in the contralateral VNC at 6 h post-UVD (P<0.05), recovering toward normal levels by 50 h.

Noah A. Russella, Arata Horiib, Paul F. Smithb, Cynthia L. Darlingtona and David K. Bilkeya, Bilateral peripheral vestibular lesions produce long-term changes in spatial learning in the rat. Journal of Vestibular Research 13 (2003) 9–16.

Abstract. In order to investigate whether bilateral peripheral vestibular lesions cause long-term impairment of spatial learning, rats were tested in a reference memory radial arm maze learning task at least 5 weeks following a bilateral labyrinthectomy (BL) or sham control lesion. All control rats reached criterion (i.e., 1 error or less, averaged across 7 trials for 3 consecutive days of training) but only 4 of the 8 BL rats had reached criterion by day 21 of the training sessions. The control rats reached criterion more quickly than the lesioned rats (Control, 7.0 ± 0.63 days, Lesioned, 15.8 ± 1.4 days, t10 = 5.84, p < 0.0001). This difference resulted from the greater number of errors made by the BL animals. However, the latency to respond was comparable as a result of the increased locomotor activity of the BL group (i.e., ‘hyperkinesis), and the overall rate of acquisition of the task, as indicated by analysis of the exponential decrease in errors over the entire training period, was not significantly different between the 2 groups. The results of this study demonstrate that BL in rats produces long-term changes in performance in a spatial reference memory task, which are not simply due to the inability to move but may relate to the way that the brain uses vestibular information to create spatial representations and determines behavioural strategies on the basis of these representations.