The morphology and gene expression of neurokinin B (NKB) neurons is altered in the human infundibular (arcuate) nucleus in association with the ovarian failure of menopause. Also, gonadotropin releasing-hormone (GnRH) mRNA is elevated and proopiomelanocortin (POMC) mRNA decreased. To determine if loss of ovarian steroids could produce comparable changes in gene expression in primates, we measured the effects of ovariectomy on NKB and GnRH in young cynomolgus monkeys. We also measured POMC gene expression, serum leptin and body weight to examine the consequences of ovariectomy on energy balance. Neurokinin B neurons in the infundibular nucleus of ovariectomized monkeys were larger, more numerous and displayed increased levels of NKB mRNA than the intact controls. Ovariectomy increased the number of neurons expressing GnRH gene transcripts. In contrast, the energy balance parameters were unchanged by ovariectomy. This study provides strong support for the hypothesis that ovarian failure contributes to the morphological changes and increased NKB and GnRH gene expression observed in postmenopausal women. We hypothesized that hypothalamic NKB neurons participate in the hypothalamic circuitry regulating LH. We determined if intracerebral infusion of a NK 3 receptor agonist alters serum LH in the ovariectomized estrogen-treated rat. A significant inhibition of serum LH was observed after senktide injection, accompanied by changes in Fos expression in medial preoptic area, arcuate, paraventricular and supraoptic nuclei. This study provides evidence that stimulation of the NK3 receptor may inhibit LH secretion via activation of hypothalamic neurons. To further investigate the role of NKB in gonadotropin regulation, we infused an antisense oligodeoxynucleotide targeted to the NKB gene in gonadectomized rats. In support of our hypothesis, the downregulation of NKB decreased serum LH by 25%. To analize the participation of the NKB receptor, NK3, we targeted an antisense to the receptor. Rats injected with the NK3 antisense exhibited no change in serum LH. Furthermore, injection of SB-222200, a NK3 antagonist, did not modify serum LH. These data suggest that NKB may regulate gonadotropin secretion through more than one receptor. Taken together, these studies provide some of the first detailed information on the relationship between NKB neurons, and the reproductive axis.

Post-menopausal women have an increased incidence of Alzheimer's disease (AD) that may be delayed in onset by estrogen replacement therapy (ERT). Estrogen has many neuroprotective and neurotrophic proclivities; therefore, its decline with menopause may leave the brain vulnerable to toxic insults stemming from disease states. Recent clinical trials investigating ERT as a treatment for AD found beneficial effects following short-term treatment that become attenuated, and possibly reversed, following longer treatment intervals. This doctoral dissertation examined the interaction of two conditions known to exist within the female AD brain: the presence of chronic neuroinflammation and either estrogen deprivation or chronic ERT. As the duration of treatment and regimen of estrogen administration may alter the effectiveness of ERT, chronic and fluctuating administration of estrogen were assessed against the behavioral, biochemical and pathological consequences of short- and long-term neuroinflammation in the female rat brain. Overall, the results suggest a strong interaction between neuroendocrine and autonomic function in the female brain with neuroinflammation. In the presence of chronic neuroinflammation, the brain differentially responds depending on the hormone status of the animal. Cognitive performance is impaired with neuroinflammation or constant estrogen; the combined occurrence of both conditions worsened performance more than either condition presented alone. However, gonadally intact females with neuroinflammation were unimpaired on the task and had approximately half the number of activated microglia. The pattern of activated microglia is unique to the female brain and highlights an interesting distribution not seen in male rats. Specifically, an elegant map of activated microglia emerges of brain areas involved in autonomic control, stress regulation and energy homeostasis. Regions showing the densest distribution of activated microglia are important autonomic relay stations that interconnect various brain regions conveying internal state information. Moreover, these regions have extensive bi-directional communication with both endocrine and immune systems, suggesting an extensive interaction occurring in the female brain capable of influencing multiple systems, including hormone secretion, sympathetic output, immune function and behavioral processes. This dissertation proposes that the interactions between these systems have important consequences for post-menopausal women with AD and are likely to underlie the varying effects seen with ERT.

Hippocampal associational and commissural pathways of the dentate gyrus include inhibitory and excitatory elements, but which inhibitory and excitatory neurons contribute to this pathway remain unclear. In addition, it is not known whether hilar mossy cells establish a recurrent excitatory network with dentate granule cells or generate lateral inhibition by activating inhibitory neurons. Improved methods for detecting neuronal markers and the retrograde tracer Fluoro-Gold (FG) were used to identify associationally and commissurally projecting neurons of the rat hippocampus and describe their three-dimensional organization. FG-positive, commissurally projecting interneurons of the dentate granule cell layer and hilus were detected in numbers greater than previously reported. FG labeling of interneurons was as high as 96% of hilar somatostatin-positive interneurons, 84% of parvalbumin-positive cells of the granule cell layer and hilus, and 33% of hilar calretinin positive cells. Whereas hilar mossy cells and CA3 pyramidal cells were FG-labeled throughout the contralateral longitudinal axis, FG-positive interneurons exhibited a relatively homotopic distribution contralaterally. In addition, retrograde transport revealed that few inhibitory interneurons were among the many retrogradely labeled hilar neurons 2.5-4.5 mm longitudinal and ipsilateral to the FG injection site. Conversely, GluR2-positive hilar mossy cells were the only with significant longitudinal associational projections. During perforant pathway stimulation in urethane-anesthetized rats, diffusion of the GABA(A) receptor antagonist bicuculline methiodide from the tip of a glass recording electrode evoked granule cell discharges and c-Fos expression in granule cells, mossy cells, and inhibitory interneurons within a ∼400 μm radius of the tip. This focally-evoked activity powerfully suppressed distant granule cell responses recorded ∼2.5-4.5 mm longitudinally. Three days after kainic acid-induced status epilepticus or prolonged perforant pathway stimulation, longitudinal inhibition was intact in rats with <40% hilar neuron loss, but was consistently abolished after extensive (>85%) hilar cell loss. These data suggest that hilar mossy cells establish surround inhibition by activating inhibitory neurons and that mossy cell loss, which is observed in the disease process of temporal lobe epilepsy, may represent a network-level mechanism underlying post-injury hippocampal dysfunction and epileptic network hyperexcitability.

Aquaporins (also known as water channels) are members of the Major Intrinsic Protein family. Ion channel function has been shown for several members of the aquaporin family and the related neurogenic gene product Big Brain. Aquaporin-1 (AQP1) is a transmembrane channel that mediates osmotically-driven water flux. Prior work demonstrated that AQP1 channels expressed in Xenopus oocytes mediate a cGMP-dependent cationic current. Based on amino acid sequence alignments with cyclic nucleotide-gated channels and cGMP-selective phosphodiesterases, I found that the efficacy of ion channel activation is decreased by mutations of AQP1 at conserved residues in the C-terminal domain (aspartate D237 and lysine K243). These data provide direct evidence for the involvement of the AQP1 carboxyl terminal domain in cGMP-mediated ion channel activation. Because the proportion of active AQP1 ion channels seen in heterologous expression systems is low, it was of fundamental importance to investigate the functional properties of this channel in a physiological context. Using rat choroid plexus, a brain tissue that secretes cerebral spinal fluid (CSF) and endogenously expresses abundant AQP1, I demonstrated the existence of native AQP1 ion channels that show properties similar to those described previously in the oocyte expression system. They mediate a cGMP-dependent cationic conductance, are blocked by cadmium, and show a single-channel conductance of 166 pS. Given the skull's rigidity, pathological increases in CSF secretion (tumors, hydrocephalus, stroke) can result in brain damage. In the choroid plexus several proteins work in concert to regulate CSF secretion. The findings presented in this dissertation are first to demonstrate that AQP1 mediates a cationic current in response to intracellular signals that regulate CSF secretion such as ANP signaling. Fluxes of water and Na⁺ across confluent choroid plexus cell monolayers showed a decreased flow rate following treatment with ANP, and Cd²⁺ reversed the inhibitory effect. These results suggest that activation and block of the AQP1-mediated ionic current may alter net fluid transport across the choroid plexus barrier, and therefore be physiologically relevant in the regulation of net fluid transport in choroid plexus. This places AQP1 as one of the important targets for clinical intervention in brain volume disorders.

This dissertation focuses on the effect of aging on the hippocampal glutamatergic system and how modulation of this system affects age-related deficits. Previous studies have shown that the function of glutamatergic system changes with age in a region-specific and receptor-specific manner. The glutamate response ratio in the dentate gyrus increases with age, which might be due to a fixed developmental process or accumulated experience over the lifespan. To address these possibilities, young rats were given one of three different treatments: standard single housing; wheel running and pair-housing; or enriched environment housing. The enrichment-treated rats learned the spatial version of the Morris swim task significantly more rapidly than did the other two groups. Neither the ratio of AMPAR- vs NMDAR-mediated field EPSP amplitudes, nor AMPAR- vs NMDAR-mediated slope conductance measured with whole-cell patch clamp recording differed significantly between groups. These results suggest that the change in the glutamate response ratio in the dentate gyrus over the lifespan is developmentally regulated. Hippocampal complex-spike cells exhibit place-specific firing, and these hippocampal place representations in young rats expand following repeated traversals of a route. The place field expansion is suggested to be important for sequence learning, and is glutamatergic system-dependent. The expansion, however, is less robust in aged rats. One question is whether the place field expansion in old rats can be restored by the application of AMPAR modulator, CX516. Injections of 35mg/kg CX516 did not result in changes in firing characteristics of CA1 pyramidal neuron ensembles, including place field expansion. Aged rats were tested using two versions of the Morris swim task. There was no drug effect on initial learning or memory of the platform location, nor on spatial learning in new visuospatial contexts after extensive training. Spatial memory in aged rats was also assessed using a radial arm maze task. CX516 facilitated memory retrieval but did not affect acquisition or consolidation processes. Thus, administration of 35mg/kg CX516 appeared to have limited effect on spatial memory performance of old rats, and did not affect place field plasticity, suggesting that this short-term plasticity may, in part, share different mechanisms with LTP.

Peripheral nerve injury can result in long-lasting, abnormal pain states referred to as neuropathic pains. These pains can result in increased sensitivity to both noxious (hyperalgesia) and non-noxious stimuli (allodynia) and are often characterized as resistant to alleviation by opioids. Neuropathies are accompanied by various neuropathological changes, including: (1) alterations in spinal neurotransmitter levels (including cholecystokinin (CCK), enkephalin and dynorphin); (2) degeneration of primary afferents; (3) formation of ectopic foci and dorsal horn sensitization; (4) abnormal sympathetic innervation and (5) sprouting of Abeta fibers to form novel synapses. The hypotheses of this dissertation are (1) information of an allodynic nature is transmitted by Abeta fibers, which do not contain opioid receptors, therefore only the post-synaptic pool of receptors is available for opioid interaction, resulting in a loss of opioid efficacy; (2) the maintenance of the neuropathic pain state is mediated by tonic activity at excitatory amino acid receptors (possibly by dynorphin) which may contribute to the loss of opioid effectiveness; and (3) increases in spinal CCK attenuate opioid effectiveness by inhibition of the activity of endogenous enkephalins. The initial hypothesis is supported by results showing that low efficacy opioids (morphine and SNC 80) are ineffective at alleviating nerve injury-induced allodynia while high efficacy opioids (DAMGO, (D-Ala2 Glu4) deltorphin and biphalin) produced a significant antiallodynic action. This strengthens the suggestion that a reduced opioid receptor pool exists for the treatment of allodynia. Additionally, selective destruction of C-fibers by capsaicin alters responses to thermal but not mechanical stimuli. These results suggests that allodynia from nerve injury is mediated by Abeta fibers, which do not possess opioid receptors. The second hypothesis is supported by the result that MK 801 and dynorphin A (1-13) antisera restore the effectiveness of morphine suggesting that increased dynorphin levels affect opioid efficacy, possibly through an NMDA mediated mechanism. Finally, blockade of CCKB receptors can also enhance the efficacy of opioids in a naltrindole reversible fashion, suggesting that CCKB blockade may increase morphine's effectiveness by increasing the availability of an endogenous delta-opioid, possibly (Leu-5) enkephalin. These findings may lead to a better understanding of the role of opioids in neuropathic pain and the development of better treatments for these conditions.

Chemical synaptic transmission requires the regulated release of neurotransmitter from synaptic vesicles stored within the presynaptic terminal. Synaptic vesicles release their contents by exocytosis, and are subsequently recycled by endocytosis and reassembly of synaptic vesicle components deposited in the plasma membrane. Previous studies have suggested that products of the Drosophila stoned gene regulate this cycling of synaptic vesicles (Petrovich et al., 1993; Andrews et al., 1996). Stoned encodes two novel proteins, stonedA and stonedB, which possess sequence motifs shared by proteins involved in intracellular vesicle traffic. Via analyses of Drosophila larval neuromuscular synapses, the work presented here provides the first direct evidence that stoned proteins regulate synaptic vesicle cycling. First, stonedA and stonedB are enriched at presynaptic terminals, and mutations of stoned decrease presynaptic levels of the stoned proteins. Second, all stoned mutations examined here disrupt neurotransmitter release, and cause mislocalization of synaptotagmin to the plasma membrane. Though this mislocalization suggests defective retrieval of synaptic vesicle components from the plasma membrane, the viable EMS-induced stnᵗˢ² and stnᶜ mutations do not decrease the supply of synaptic vesicles or alter the assembly of morphologically normal vesicles. Thus, impaired neurotransmitter release at stnᵗˢ² and stnᶜ synapses either arises from subtle defects of synaptic vesicle recycling, or indicates a role for the stoned proteins in synaptic vesicle exocytosis. The stn⁸ᴾ¹ mutation severely reduces synaptic transmission, decreases synaptic vesicle density and increases synaptic vesicle size. Thus, altered neurotransmitter release at stn⁸ᴾ¹ synapses certainly arises in part from defects in synaptic vesicle endocytosis. Unexpectedly, all three stoned mutants show overgrowth of the presynaptic terminal. In the stn⁸ᴾ¹ mutant, terminals have a large number of bud-like satellite boutons, also observed in Drosophila overexpressing the Amyloid Precursor Protein, a cell surface protein implicated in human Alzheimer's Disease. Thus, altered presynaptic structure in stoned mutants might indicate impaired endocytosis of cell-cell signaling molecules that regulate synapse growth. Overall, these data establish that stoned proteins regulate the trafficking of synaptic vesicles, and suggest that stoned proteins additionally regulate cellular trafficking pathways that influence synaptic plasticity.

Despite a wealth of information regarding visual processing in flies, little is known regarding their pattern discrimination abilities and the neural correlates of pattern recognition. The work presented here provides evidence of learned visual discriminations of complex visual patterns in the blowfly, Phaenicia sericata. A learning and memory assay was developed to determine the pattern-orientation discrimination ability of blowflies. Individual flies were trained to discriminate between pairs of visual patterns that differed in the orientation of their composite lines. During training and subsequent testing trials flies exhibited a preference for the previously-rewarded visual stimulus. Flies learned to discriminate between horizontal and vertical gratings, +45° and -45° gratings, and vertical and +5° gratings. Among four possible underlying mechanisms used to discriminate between these sets of patterns, hypothetical orientation-selective neurons are uniquely capable of discriminating between all sets of discriminated patterns. One previous theory of insect vision suggests that in order for an insect to recognize previously learned visual images, the insect must align the current retinal input with the region of the retina with which the pattern was first viewed and learned (Wehner, 1981; Dill et al., 1993). To address this theory of retinotopic matching, a detailed analysis was made of fly behavior during the discrimination process. Both the spatial structure of the approaches to the visual cues and the range of body orientations used by individual flies demonstrated that Phaenicia approaches the visual cue from an unique vantage point from trial to trial and, therefore, retinotopic matching is not necessary for the recognition of pattern orientation by blowflies. To test for the existence of orientation-selective neurons in Phaenicia, the lobula neuropil was impaled for intracellular recording. Two neurons exhibited directional motion preferences indicative of a possible role in the detection of expanding, oblique edges during approach to visual cues. Two additional neurons with combined responses to both directional motion and the orientation of the stimulus were recorded. These data support the hypothesis that the dipteran lobula processes information regarding the orientation of visual stimuli.

Shortly following the advent of intracellular (IC) recording from spinal cord (SC) motoneurons (MNs) in the anesthetized cat, Eccles (1957) proposed that MNs behave passively in response to synaptic input. It is now known, however, that repetitive MN discharge is subject to the influence of endogenous neurotransmitters and neuromodulators that alter sub- and/or supra-threshold ionic conductances. Much of the literature in this field is anecdotal and focussed on ionic mechanisms. Further research is therefore required on the robustness of neuromodulatory effects, their significance during natural and fictive movements, and their generalization across vertebrate species. Accordingly, the purpose of this study was to quantitate the effects of neuromodulation on MN properties determined from the SC slice of the adult turtle. The present work is divided into four parts. The first (Chapter 2) summarizes the literature on MN behavior as determined from a variety of vertebrate preparations. The second (Chapter 3) provides an evaluation of the robustness of the electrophysiological measurements made from turtle SC MNs and compares them to analogous results from the lamprey and cat. The third part (Chapter 4) reports on the MNs' responses to three excitatory and one inhibitory neuromodulator. The MN population was divided into two groups on the basis of their propensity to generate plateau potentials (PPs). In PP MNs, the slope of the stimulus current-spike frequency relation was flattened to an extent comparable to recent findings in the decerebrate cat preparation. The fourth component of the study (Chapter 5) provides a comparison of MN behavior in a variety of preparations across vertebrate species, with particular emphasis on the functional efficacy of the PP. In summary, the present work has provided a new opening in the study of neuromodulation on vertebrate spinal MN properties by quantifying the effects of such modulation. In addition, the work has added further evidence supporting an evolutionary conservation of MN properties across vertebrates, with a particular emphasis on the functional significance of the PP as a key determinant of MN discharge.

During slow-wave sleep (SWS), traces of neuronal activity patterns from preceding behavior can be observed in rat hippocampus and neocortex. The spontaneous reactivation of these patterns is manifested as the reinstatement of the distribution of pairwise firing rate correlations within a population of simultaneously recorded neurons. The effects of behavioral state (quiet wakefulness, SWS and REM), interactions between two successive spatial experiences, and global modulation during 200 Hz EEG "ripples" on pattern reinstatement were studied in CA1 pyramidal cell population recordings. Pairwise firing rate correlations during often repeated experiences accounted for a significant proportion of the variance in these interactions in subsequent SWS or quiet wakefulness and, to a lesser degree, during SWS prior to the experience on a given day. The latter effect was absent for novel experiences, suggesting that a persistent memory trace develops with experience. Pattern reinstatement was strongest during sharp wave-ripple oscillations, suggesting that these events may reflect system convergence onto attractor states corresponding to previous experiences. When two different experiences occurred in succession, the statistically independent effects of both were evident in subsequent SWS. Thus, the patterns of neural activity reemerge spontaneously, and in an interleaved fashion, and do not necessarily reflect persistence of an active memory (i.e., reverberation). Firing rate correlations during REM sleep were not related to the preceding familiar experience, possibly as a consequence of trace decay during the intervening SWS. REM episodes also did not detectably influence the correlation structure in subsequent SWS, suggesting a lack of strengthening of memory traces during REM sleep, at least in the case of familiar experiences.