abstract: Development of the cerebral cortex requires the complex integration of extracellular stimuli to affect changes in gene expression. Trophic stimulation activates specialized intracellular signaling cascades to instruct processes necessary for the elaborate cellular diversity, architecture, and function of the cortex. The canonical RAS/RAF/MEK/ERK (ERK/MAPK) cascade is a ubiquitously expressed kinase pathway that regulates crucial aspects of neurodevelopment. Mutations in the ERK/MAPK pathway or its regulators give rise to neurodevelopmental syndromes termed the “RASopathies.” RASopathy individuals present with neurological symptoms that include intellectual disability, ADHD, and seizures. The precise cellular mechanisms that drive neurological impairments in RASopathy individuals remain unclear. In this thesis, I aimed to 1) address how RASopathy mutations affect neurodevelopment, 2) elucidate fundamental requirements of ERK/MAPK in GABAergic circuits, and 3) determine how aberrant ERK/MAPK signaling disrupts GABAergic development. Here, I show that a Noonan Syndrome-linked gain-of-function mutation Raf1L613V, drives modest changes in astrocyte and oligodendrocyte progenitor cell (OPC) density in the mouse cortex and hippocampus. Raf1L613V mutant mice exhibited enhanced performance in hippocampal-dependent spatial reference and working memory and amygdala-dependent fear learning tasks. However, we observed normal perineuronal net (PNN) accumulation around mutant parvalbumin-expressing (PV) interneurons. Though PV-interneurons were minimally affected by the Raf1L613V mutation, other RASopathy mutations converge on aberrant GABAergic development as a mediator of neurological dysfunction. I therefore hypothesized interneuron expression of the constitutively active Mek1S217/221E (caMek1) mutation would be sufficient to perturb GABAergic circuit development. Interestingly, the caMek1 mutation selectively disrupted crucial PV-interneuron developmental processes. During embryogenesis, I detected expression of cleaved-caspase 3 (CC3) in the medial ganglionic eminence (MGE). Interestingly, adult mutant cortices displayed a selective 50% reduction in PV-expressing interneurons, but not other interneuron subtypes. PV-interneuron loss was associated with seizure-like activity in mutants and coincided with reduced perisomatic synapses. Mature mutant PV-interneurons exhibited somal hypertrophy and a substantial increase in PNN accumulation. Aberrant GABAergic development culminated in reduced behavioral response inhibition, a process linked to ADHD-like behaviors. Collectively, these data provide insight into the mechanistic underpinnings of RASopathy neuropathology and suggest that modulation of GABAergic circuits may be an effective therapeutic option for RASopathy individuals. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2019

Dopamine (DA) transmission is a key player in the rewarding aspects of ethanol as well as ethanol dependence. The current dogma is that DA transmission is increased during ethanol via the inhibition of ventral tegmental area (VTA) GABA neurons and that excitation of VTA GABA neurons during withdrawal results in decreased DA transmission. Microglia, the major neuroimmune effector in the brain, may be a key mediator in this process by releasing cytokines following activation. We evaluated the effect of ethanol on cytokine concentrations in the VTA and NAc using a cytometric bead array, and found that low dose ethanol (1.0 g/kg) decreased interleukin (IL)-10 levels, but high dose ethanol increased IL-10 levels (4.0 g/kg). We also used standard cell-attached mode electrophysiological techniques to evaluate the effects of select cytokines on VTA neuron firing rate in vitro. We found no change in firing rate in response to IL-6, but an increase in firing rate in VTA DA neurons response to IL-10. Consistent with the changes in firing rate, optically-evoked IPSCs were also found to be decreased in response to IL-10. Ex vivo voltammetry and in vivo microdialysis were done to determine whether IL-10 can directly result in an increase in DA release. Although ex vivo voltammetry showed no change in DA release, IL-10 increased DA release in vivo. These findings suggest that the rewarding and/or addictive effects of ethanol are mediated by cytokines, specifically the anti-inflammatory cytokine IL-10.

Patch-clamp recordings in whole-cell configuration were made from ventrolateral medulla neurons of brainstem slices from 8-12-day-old rats. 5- Hydroxytryptamine (3-30 μM) concentration-dependently suppressed excitatory and inhibitory postsynaptic currents evoked by focal stimulation. An augmentation of inhibitory synaptic currents by 5-hydroxytryptamine was noted in a small number of neurons. 5-Hydroxytryptamine depressed synaptic currents with or without causing a significant change in holding currents and membrane conductances; the inward or outward currents induced by exogenously applied glutamate or GABA/glycine were also not significantly changed by 5- hydroxytryptamine. In paired-pulse paradigms designed to evaluate a presynaptic site of action, 5-hydroxytryptamine suppressed synaptic currents but enhanced the paired-pulse facilitation. 5-Hydroxytryptamine reduced the frequency of miniature excitatory postsynaptic currents without significantly affecting the amplitude. 5-Carboxamidotryptamine, 8-hydroxy-2(di-n- propylamino)tetralin, sumatriptan and N-(3-trifluoromethylphenyl)piperazine which exhibit 5-hydroxytryptamine1 receptor agonist activity, depressed synaptic currents with different potencies, with 5-carboxamidotryptamine being the most potent. The non-selective 5-hydroxytryptamine1 receptor antagonist pindolol attenuated the presynaptic effect of 5-hydroxytryptamine, whereas the 5-hydroxytryptamine(1A) antagonist pindobind-5- hydroxytryptamine(1A) and 5-hydroxytryptamine2 receptor antagonist ketanserin were ineffective. Our results indicate that 5-hydroxytryptamine suppressed synaptic transmission in ventrolateral medulla neurons by activating presynaptic 5-hydroxytryptamine1 receptors, probably the 5- hydroxytryptamine(1B)/5-hydroxytryptamine(1D) subtype. In addition, 5- hydroxytryptamine augmented inhibitory synaptic currents in a small number of neurons the site and mechanism of this potentiating action are not known.

To study the influence of the central noradrenergic system on sensitivity to sedative-hypnotic effects mediated by the aminobutyric acid (GABA) system, intact rats were contrasted with rats in which noradrenergic nerves were largely destroyed shortly after birth with the neurotoxin DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine; 50 mg/kg sc x2, P1 and P3]. At 10 weeks, loss of the righting reflex (LORR) was used as an index to study the acute sedative-hypnotic effects of phenobarbital (100 mg/kg ip) and ethanol (4 g/kg ip, 25% v/v). Additionally, GABA concentration in the medial prefrontal cortex (PFC), hippocampus, cerebellum and brainstem was estimated by an HPLC/ED method. Neonatal DSP-4 treatment diminished the sedative-hypnotic effects of both phenobarbital and ethanol in adult rats. While the endogenous GABA content in the PFC, hippocampus, brainstem and cerebellum of DSP-4-treated rats was not altered, phenobarbital significantly decreased GABA content of both intact and DSP-4-lesioned rats by ∼40% in the hippocampus and by ∼20% in other brain regions at 1 h. Ethanol reduced GABA content by ∼15-30% but only in the hippocampus and brainstem of both intact and lesioned rats. These findings indicate that the noradrenergic system exerts a prominent influence on sedative-hypnotics acting via GABAergic systems in the brain without directly altering GABA levels in the brain.

No description available.

Although it is well-established that animals will self-stimulate electric current to various diverse brain structures, the neural substrate of brain stimulation reward (BSR) has eluded identification since its discovery more than a half-century ago. We show that GABA neurons in the midbrain, hypothalamus and thalamus express connexin-36 (Cx36) gap junctions and couple electrically with dopamine application or by stimulation of the internal capsule (IC), which also supports self-stimulation. The threshold for responding for self-stimulation of the IC is the threshold for coupling between these GABA neurons, the degree of responding for IC ICSS is proportional to the magnitude of electrical coupling between these GABA neurons, and GJ blockers, including the Cx36 blocker mefloquine, increase the threshold for IC self-stimulation without affecting performance. Thus, electrical coupling between this network of GABA neurons fits the prevailing model for the elusive integrator of BSR.

The purpose of this project was to investigate the mechanism of action of acupuncture on a critical neural substrate involved in alcoholism. Specifically, this study evaluated the effects of stimulation of the acupuncture Shenmen (HT7) point on inhibitory GABA neurons in the ventral tegmental area (VTA), a midbrain structure implicated in drug and alcohol abuse, and ethanol self-administration. In addition, the role of opioid receptors (ORs) in ethanol and acupuncture effects was explored. Using electrophysiological methods in mature rats, we evaluated the effects of HT7 stimulation and opioid antagonists on the VTA GABA neuron firing rate. With behavioral paradigms, we also assessed those effects on ethanol self-administration, using a modification of the sucrose fading procedure. We found that HT7 stimulation produced a biphasic modulation of VTA GABA neuron firing rate characterized by transient enhancement at the onset of stimulation followed by a prolonged inhibition and subsequent recovery in 5 min. HT7 stimulation blocked the typical suppression of VTA GABA neuron firing rate produced by a moderately intoxicating dose of ethanol. The late inhibition produced by HT7 stimulation as well as HT7 reversal of ethanol's effects on GABA neuron firing rate was blocked by the non-selective opioid receptor antagonist, naloxoneIn addition, HT7 acupuncture reduced ethanol self-administration without affecting sucrose consumption. More important, systemic administration of the δ-opioid receptor (DOR) antagonist, naltrindole blocked ethanol suppression of VTA GABA neuron firing rate and significantly reduced ethanol self-administration without affecting sucrose consumption. These findings suggest that DOR-mediated opioid modulation of VTA GABA neurons may be related to the role of acupuncture in modulating mesolimbic DA release and suppressing the reinforcing effects of ethanol. We confirmed that acupuncture stimulation may have a significant impact on the inhibitory neuron activity in the VTA and that acupuncture may serve as an effective adjunct to OR antagonist therapy for alcoholism.

The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system leads to the rewarding properties of alcohol and nicotine (NIC). The mesolimbic DA system consists of DA neurons in the midbrain ventral tegmental area (VTA) that innervate the nucleus accumbens (NAc). DA neurotransmission is regulated by inhibitory VTA GABA neurons, whose excitability is a net effect of glutamate (GLU) and GABA neurotransmission that are modulated by NIC cholinergic receptors (nAChRs) on afferent terminals. We have previously demonstrated that VTA GABA neurons are excited by low-dose ethanol but are inhibited by moderate to high-dose ethanol, and they adapt to chronic ethanol, evincing marked hyperexcitability during withdrawal. The aim of this study was to evaluate the role of α6 nAChRs in ethanol effects on VTA GABA and DA neurons. In order to more conclusively demonstrate the role of α6 nAChRs in alcohol modulation in the VTA, we profiled the pharmacological interactions between ethanol and α6 nAChRs using recombinant α6 nAChRs in human epithelia (SH-EP1) cells and evaluated the effects of α6 nAChR antagonists on ethanol inhibition of GABA-mediated synaptic responses in dissociated GABA neurons of the VTA by recording mIPSCs; and assessed the effects of α6 nAChR antagonists on ethanol inhibition of VTA neurons, via eIPSCs on GABA neurons, sIPSCs on GABA neurons, and firing rate of DA neurons. We found that ethanol enhanced NIC currents in SH-EP1 cells via α6 nAChRs. Electrophysiology studies showed that superfusion of ethanol (5-30 mM) enhanced the frequency and amplitude of mIPSCs recorded in acutely dissociated VTA GABA neurons from GAD-GFP mice. Furthermore, the α6 nAChR antagonist α-conotoxin P1A (10 nM) prevented the ethanol-induced changes in mIPSC. In support, eIPSC experiments demonstrated that low doses of ethanol (1-5 mM) enhanced eIPSC peaks and decreased paired-pulse ratio, suggesting a presynaptic effect with ethanol. Alpha-conotoxin MII (α-CTX MII) blocked ethanol's effects on eIPSCs. This effect on VTA GABA neurons was also seen in sIPSCs, as ethanol decreased GABA firing rate. Similarly, the inhibition caused by ethanol was prevented by α-conotoxin P1A (10 nM). Additionally, CPP studies showed that α6 KO mice and WT mice treated with MEC, a non-competitive, non-α7 antagonist, did not show a preference for EtOH compartments that was found in WT mice. Taken together, these studies indicate that ethanol is acting through α6 nAChRs on GABA terminals to enhance GABA release, suggesting a possible mechanism of action of alcohol and nicotine co-dependence. Through these studies conducted to understand the role of α6 nAChRs in ethanol modulation, we hope to further outline how alcohol alters brain activity so that we can ultimately facilitate the development of therapies/medications for the treatment of alcoholism.

No description available.

Spatial navigation in humans correlates with activity of cells in hippocampus that respond when we traverse specific locations in our environment. Hippocampal pyramidal cells in rodents called "place cells" may contribute to episodic memory by encoding location in physical space. Place cells display plasticity by "remapping" or altering their firing rates and patterns of activity in response to changes in spatial environment. Impaired remapping may underlie age-related deficits in spatial memory tasks. Using in vivo high-density electrophysiology to record place cell activity in awake, behaving rats, we tested the hypothesis that CA3 neuron hyperactivity in aged animals could be normalized by pharmacotherapy. Results show that acute, systemic administration of low dose levetiracetam and sodium valproate ameliorates deficits in the aged hippocampal network by reducing firing rates, decreasing place field area, and increasing the spatial selectivity of CA3 place cells. We then tested the hypothesis that place cell activity, field area, and spatial selectivity may be an indicator for therapeutic enhancement of spatial memory in young adult rats. The results demonstrate that α5IA enhances hippocampal-dependent spatial memory as measured by the location novelty recognition task in rats, consistent with the previously established action of α5IA as an enhancer of spatial memory in the water maze test. Electrophysiological recordings on the same animals carried out in parallel demonstrate that α5IA increases place cell firing rates, reduces field area, and increases spatial selectivity. Together, these results suggest that reducing place field area and enhancing spatial selectivity correlate with the age-independent therapeutic improvement of spatial memory. The increase in place cell firing rates by α5IA likely results from its known action as a negative allosteric modulator of α5-subunit-containing receptors (α), which are located extrasynaptically at the base of dendritic spines on CA1 and CA3 pyramidal cells. Thus, to potentially target extrasynaptic tonic inhibition in the hippocampus, we synthesized and validated two α specific miRNAs as a platform for future attempts to improve spatial memory in young adult and aging animals via molecular genetics.