1. Whole-cell patch recordings were made from substantia gelatinosa (SG) neurons in transverse lumbar spinal cord slices of 15- to 30-day-old rats. 2. Endomorphin 1 (EM-1) or EM-2 (≤ 10 μM) hyperpolarized or induced an outward current in 26 of the 66 SG neurons. The I-V relationship showed that the peptide activates an inwardly rectifying K + current. 3. EM-1 or EM-2 (0.3-10 μM) suppressed short-latency excitatory postsynaptic currents (EPSCs) and long-latency inhibitory postsynaptic currents (IPSCs) in nearly all SG neurons tested or short-latency IPSCs in six of the 10 SG neurons. [Met 5] enkephalin or [D-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin (DAMGO) (1-10 μM) depressed EPSCs and IPSCs. EM-1 or EM-2 depressed synaptic responses without causing a significant change in holding currents or inward currents induced by glutamate. 4. Glutamate also evoked a short-latency outward current in five SG neurons or a biphasic current in two neurons; the outward current was blocked by tetrodotoxin (TTX, 0.3 μM) or bicuculline (10 μM). EM-1 or DAMGO (1 or 5 μM) attenuated the glutamate-evoked outward or biphasic currents in four of the seven SG neurons. 5. EM-1 (1 μM) reduced the frequency, but not the amplitude of miniature EPSCs or miniature IPSCs. 6. Naloxone (1 μM) or the selective μ-opioid receptor antagonist β-funaltrexamine (β-FNA, 25 μM) antagonized the action of EM; EM-induced hyperpolarizations persisted in the presence of the κ-opioid receptor antagonist (nor-binaltorphimine dihydrochloride, 1 μM) and/or σ-opioid receptor antagonist (naltrindole hydrochloride, 1 μM). 7. It may be concluded that EM acting on μ-opioid receptors hyperpolarizes a population of SG neurons by activating an inwardly rectifying K + current, and attenuates excitatory and inhibitory synaptic currents evoked in a population of SG neurons, probably by a presynaptic site of action.

Indiana University-Purdue University Indianapolis (IUPUI) / Serotonin (5-HT) is heavily implicated in severe anxiety and trauma-related disor-ders, such as panic and post-traumatic stress disorders. Overall, site-specific pharmacolog-ical manipulations show that while 5-HT enhances anxiety-associated/avoidance behaviors in the amygdala, 5-HT inhibits panic-associated escape behaviors in the perifornical hypo-thalamus region (PeFR). Yet, our understanding of how specific serotonergic networks and co-transmitters regulate these conditions, but also other aspects of innate panic (e.g., car-dioexcitation or thermal response that occur during a flight or escape response) or condi-tioned fear behaviors is still elusive. Therefore, utilizing circuit-based gain- and loss-of-function approaches to selectively manipulate amygdala- and PeFR-projecting sero-tonergic systems, we hypothesize that specific serotonergic networks projecting to the amygdala and PeFR respectively enhance conditioned fear responses and attenuate innate panic-associated behaviors and physiological responses. There are two main chapters in this dissertation. In Chapter III, retrograde tracing revealed that the amygdala-projecting neurons from dorsal Raphe (DR) were almost exclusively serotonergic (92-95%) concen-trated in the dorsal/ventral (DRD/DRV) DR, with few non-serotonergic neurons. While selective lesioning of this network with saporin toxin (SAP) facilitated the extinction of conditioned fear behavior, selective optogenetic activation of amygdala-projecting DRD/DRV cell bodies using intersectional genetics reduced extinction of conditioned fear behavior and enhanced anxiety avoidance. In Chapter IV, retrograde tracing showed that the PeFR was innervated by equally selective serotonergic networks concentrated in the lateral wings DR (lwDR) and median Raphe (MR). Contrasting with the results from the amygdala-innervating 5-HT system, lesioning the PeFR-projecting serotonergic network from lwDR/MR was accompanied by reduced extinction of conditioned fear behavior, in-creased anxiety avoidance, and increased CO2-induced panic (elevated escape responses and enhanced cardioexcitation). Conversely, selective activation of lwDR/MR serotonergic terminals in the PeFR decreased anxiety-associated behaviors; inhibited CO2-induced panic, and induced unconditioned and conditioned place preferences. The circuit-based ap-proach data presented here show that amygdala- and PeFR-projecting 5-HT neurons com-prise distinct circuits underlying opposite roles enhancing anxiety/fear responses in the amygdala and dampening fear/panic responses in the PeFR. The identification of distinct circuits controlling anxiety, fear, and panic responses is a fundamental step towards the development of more effective therapies for psychiatric conditions such as anxiety and trauma-related disorders. / 2021-11-04

Los seres humanos están continuamente expuestos a disruptores endocrinos como el bisfenol A (BPA). El BPA es un componente de los plásticos de policarbonato y resinas epoxi. Se encuentra en muchos productos de uso cotidiano, como envases de comida o el papel térmico de los tickets de la compra. El BPA tiene actividad estrogénica y puede afectar a diversos sistemas del organismo. Se sabe que el 17-β-estradiol aumenta la nocicepción a través de la modulación de diferentes canales iónicos y receptores. Sin embargo, no se conoce el papel del BPA en los mecanismos de nocicepción. En este trabajo, se realizó un estudio para examinar los efectos de una dosis ambientalmente relevante de BPA (1 nM) en la función y expresión de los canales de sodio dependientes de voltaje (Nav) de las neuronas del ganglio de la raíz dorsal (DRG) y determinar el mecanismo de acción del BPA a través de los receptores de estrógenos (ER). Para ello, se llevaron a cabo experimentos electrofisiológicos para analizar los parámetros biofísicos de los canales NaV. Las neuronas tratadas con BPA mostraron un aumento de la corriente de rampa de los canales Nav1.7y de la frecuencia de disparo de potenciales de acción. También se realizó un ensayo de expresión en el que se vio que el BPA no modificaba la expresión de los canales de Nav de las neuronas de los DRGs. Por otra parte, se realizó un experimento de comportamiento para evaluar las respuestas al dolor térmico. Los ratones tratados con inyecciones subdérmicas de BPA (50 μg/kg/día) mostraban un tiempo de respuesta al estímulo menor que los ratones control. Para determinar el mecanismo a través del cual el BPA modificaba las propiedades biofísicas del canal Nav1.7, se utilizaron agonistas y antagonistas de los receptores de estrógenos ERα y ERβ, así como ratones knockout para ERβ. Las neuronas fueron incubadas durante 24 h con 1 mM PPT (agonista de ERα), que ejercía el mismo efecto que el BPA sobre la corriente de rampa. Por otra parte, la acción del BPA fue bloqueada con 100 nM MPP (antagonista de ERα). Además, se observó que la potenciación de la corriente de rampa producida por el BPA se veía disminuida al usar 100 nM wortmanina (inhibidor de PI3K). En conclusión, este estudio sugiere que el BPA altera la corriente de rampa del canal NaV1.7 y aumenta la excitabilidad en neuronas de los DRGs a través de la vía de ERα y PI3K.

No description available.

FFifteen to thirty-five percent of the population in the United States experience tinnitus, a subjective "ringing in the ears". Up to 10% of tinnitus patients report their symptoms are severe and disabling. Tinnitus was induced in FBN rats using 116 dB (SPL) unilateral octave-band sound exposures centered at 16 kHz for one hour in an anesthetized preparation. Rats were assessed behaviorally by an operant conditioning paradigm as well as a gap detection method to verify the development of tinnitus. Both young (7 mos.) and aged (30 mos.) sound exposed rats showed significant elevated auditory brainstem-evoked response (ABR) thresholds for clix and all tested frequencies immediately after the sound exposure. Eighty days post-exposure, ABR thresholds for the young exposed rats were significantly close to the initial young control values while aged exposed rats showed residual thresholds shifts relative to aged controls. Sixteen weeks following sound exposure, young exposed rats showed significantly reduced gap detection at 24 and 32 kHz, suggestive of high frequency tinnitus. Aged exposed animals showed significant tinnitus-related behavioral changes near 10 kHz by both behavior methods. Message and protein levels of &alpha1-3 glycine receptor subunits (GlyRs), gephyrin, BDNF and its receptor TrkB were assessed in dorsal cochlear nucleus (DCN) fusiform cells 4 months post exposure utilizing quantitative in situ hybridization and immunocytochemistry. Young exposed rats showed significant decreases of GlyR &alpha1 protein at middle and high frequency regions in DCN unlike the contrasting increase of their message levels. Aged exposed rats showed higher &alpha1 subunit protein levels in the same high and middle DCN frequency regions. The GlyR anchoring protein, gephyrin, was significantly increased in both young and aged exposed rats, suggesting an intracellular receptor trafficking change following acoustic trauma. BDNF and TrkB were also increased over fusiform cells in both young and aged exposed rats. [3H] strychnine binding was used to evaluate DCN GlyR pharmacology and function following sound exposure. The age-related decrease in GlyR α1 protein was reflected in the significant age-related down-regulation of GlyR (Bmax). Tinnitus-related changes in GlyR &alpha1 protein level was reflected in the decline of the GlyR (Bmax) in young exposed rats and up-regulated GlyRs in aged exposed animals. The GlyRs in DCN of young exposed animals also demonstrated an increase in affinity, further suggesting a post-exposure receptor composition change. These findings suggest that both aging and/or sound exposure/tinnitus are associated with GlyR changes capable of altering alter the output of the DCN. Detailed characterization of these GlyR modifications could advance the development of novel selective drugs for tinnitus and age-related hearing loss.

The hippocampus is a brain region that can undergo tremendous plasticity in adulthood. The hippocampus is related to the formation of spatial memories in birds and mammals. In birds, plasticity in the hippocampus occurs when formation of such memories is directly relevant to survival or reproduction, such as for breeding or food caching. In reptiles, the homologues to the hippocampus are the dorsal and medial cortices (DC and MC). In several lizard, snake and turtle species, these structures have been related to spatial memory. Experimental investigations indicate that differences in DC volume are related to space use associated with differing foraging ecologies. Differences in MC volume have been associated with territory size-based mate acquisition strategies. Furthermore, territory size has previously been correlated with plasma testosterone (T) levels. Therefore, I hypothesized that neuroplasticity within the MC/DC is controlled by demands on spatial navigation and seasonal differences and that these changes may involve the action of T. During two experimental trials, male Western Fence Lizards (Sceloporus occidentalis) were placed into either large or small semi-natural enclosures and allowed to interact with a female and intruder males over the span of seven weeks. One trial was performed during the spring breeding season and the other during the summer non breeding season, to examine seasonal differences in plasticity. Blood samples were collected at initial time of capture and before sacrifice to measure plasma T. Immunostaining for doublecortin was used to determine the density of immature neurons in each region, and cresyl violet staining allowed for volume measurements of specific regions. MC cell layer neurogenesis was higher in lizards placed in large enclosures than those in small enclosures and higher in the summer than in the spring. DC volume was smaller in lizards held in large enclosures than those in small enclosures. The decreased DC volume seen lizards held in large enclosures may indicate a cost to the increased neurogenesis in the MC of lizards in the same enclosures. These results indicate a possible trade-off between DC volume and MC neurogenesis that allows for switching between the ability to solve novel spatial tasks using the DC while storing a cognitive map in the MC. During the spring, T had no relationship with MC volume, while during the summer this was negative, so effects of T on the MC may be seasonal.

Drosophila melanogaster has a tubular heart called the dorsal vessel, which is composed of contractile cardiomyocytes and hemolymph filtering pericardial cells. During larval development the dorsal vessel (heart) grows in size, and the luminal space inside the heart expands, however it has not been clear which cells are responsible for laying the extracellular matrix (ECM) during this expansion. Hemocytes (white blood cells), pericardial cells and cells of the fat body are candidate cell types that may secrete ECM for assembly during the growth of the heart lumen. With gene knock-down techniques we are exploring whether hemocytes participate in assembly of the heart ECM at this location. Additionally, studies of fluorescently tagged hemocytes in intact larvae reveal that hemocytes aggregate around pericardial cells of the dorsal vessel in 3rd instars. Confocal studies of dissected larval hearts indicate that hemocytes aggregate within infoldings of basement membrane associated with pericardial cells. Hemocyte-pericardial cell association could indicate that hemocytes take up proteins that are produced by pericardial cells and deliver them to other locations or that there might be a previously unidentified hematopoietic site at the Drosophila larval heart. / Thesis / Master of Science (MSc)

<p> Osteoarthritis (OA) is the most common arthritis, and the second most common diagnosis leading to disability. While loss of joint function is disabling, patients report that the greatest disabler of OA is the pain. Unfortunately, OA pain remains an unmet medical need. Numerous mechanisms have been proposed for the pathogenesis of OA pain. However, none of these mechanisms has led to satisfactory evidence-based treatment for OA pain. There is a critical need to address the mechanisms for OA pain due to the aging demographics and the prevalence of OA in older adults. This thesis project was aimed to study neural mechanisms for OA pain. The general hypothesis was that the pain of OA arises as a result of phenotypic changes in primary sensory neurons, especially in larger diameter A-fiber neurons. In vivo intracellular recordings were used to determine changes in specific populations of DRG neuron in a surgical knee derangement model of OA in the rat. It was found that AB-fiber low threshold mechanoreceptors, particularly muscle spindle afferents underwent significant changes (including changes in action potential configurations and in responses to repetitive stimulation) one month following the model induction when histopathological changes of the knee joint and the nocifensive behaviors of the affected lower limb favor OA. Nociceptors, including C-, As- and AB-fiber neurons remained largely unchanged at one month OA. AB-fiber high threshold mechanoreceptors exhibited significant changes at two month OA, a later phase during the progression of OA. The data demonstrate that distinct populations of dorsal root ganglia neuron are altered during the progression of OA, which might be the neuronal basis for clinical presentations of sensory deficit in OA including pain and loss of proprioception. The data also suggest that the pain in OA might be a form of neuropathic pain. </p> / Thesis / Doctor of Philosophy (PhD)

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No description available.