Projection neurons of nucleus accumbens (NAC) of the rat were identified by either antidromic activation from stimulation of midbrain ventral tegmental area-substantia nigra (VTA-SN) regions, or by tracing axons of intracellularly labeled NAC neurons into the ventral pallidum. The morphology of these NAC projection neurons were determined to be medium spiny neurons similar to those identified in the caudate-putamen.

1. We studied the effect of intracellular angiotensin II (Ang II) and related peptides on rat aortic contraction, whether this effect is pharmacologically distinguishable from that induced by extracellular stimulation, and determined the Ca2+ source involved. 2. Compounds were delivered into the cytoplasm of de-endothelized aorta rings using multilamellar liposomes. Contractions were normalized to the maximum obtained with phenylephrine (10-5 M). 3. Intracellular administration of Ang II (incorporation range: 0.01-300 nmol mg-1) resulted in a dose-dependent contraction, insensitive to extracellular administration (10-6 M) of the AT1 receptor antagonist CV11947, the AT2 receptor antagonist PD 123319, or the non-selective AT receptor antagonist and partial agonist saralasin ([Sar1,Val5,Ala8]-Ang II (P < 0.05). 4. Intracellular administration of CV11947 or PD 123319 right shifted the dose-response curve about 1000 fold or 20 fold, respectively. PD 123319 was only effective if less than 30 nmol mg-1 Ang II was incorporated. 5. Contraction was partially desensitized to a second intracellular Ang II addition after 45 min (P < 0.05). 6. Intracellular administration of Ang I and saralasin also induced contraction (P < 0.05). Both responses were sensitive to intracellular CV11947 (P < 0.05), but insensitive to PD 123319. The response to Ang I was independent of intracellular captopril. 7. Contraction induced by extracellular application of Ang II and of Ang I was abolished by extracellular pre-treatment with saralasin or CV11947 (P < 0.05), but not with PD 123319. Extracellular saralasin induced no contraction. 8. Intracellular Ang II induced contraction was not affected by pre-treatment with heparin filled liposomes, but completely abolished in Ca2+-free external medium. 9. These results support the existence of an intracellular binding site for Ang II in rat aorta. Intracellular stimulation induces contraction dependent on Ca2+-influx but not on Ins(1,4,5)P3 mediated release from intracellular Ca2+-stores. Intracellular Ang I and saralasin induce contraction, possibly via the same binding site. Pharmacological properties of this putative intracellular receptor are clearly different from extracellular stimulated AT1 receptors or intracellular angiotensin receptors postulated in other tissue.

Hepatocytes exhibit a regulatory volume decrease (RVD) during hypotonic shock, which comprises loss of intracellular K+ and Cl- accompanied by hyperpolarization of transmembrane potential (Vm) due to an increase in membrane K+ conductance, (GK). To examine hepatocyte K+ homeostasis during RVD, double-barrel, K+-selective microelectrodes were used to measure changes in steady-state intracellular K+ activity (aKi) and Vm during hyposmotic stress. Cell water volume change was evaluated by measuring changes in intracellular tetramethylammonium (TMA+). Liver slices were superfused with modified Krebs physiological salt solution. Hyposmolality (0.8×300 mosm) was created by a 50 m m step-decrease of external sucrose concentration. Hepatocyte Vm hyperpolarized by 19 mV from -27 ± 1 to -46 ± 1 mV and aKidecreased by 14% from 91 ± 4 to 78 ± 4 m m when slices were exposed to hyposmotic stress for 4-5 min. Both Vm and aKireturned to control level after restoring isosmotic solution. In paired measurements, hypotonic stress induced similar changes in Vm and aKiboth control and added ouabain (1 m m) conditions, and these values returned to their control level after the osmotic stress. In another paired measurement, hypotonic shock first induced an 18-mV increase in Vm and a 15% decrease in aKiin control condition. After loading hepatocytes with TMA+, the same hypotonic shock induced a 14-mV increase in Vm and a 14% decrease in aTMAi. This accounted for a 17% increase of intracellular water volume, which was identical to the cell water volume change obtained when aKiwas used as the marker. Nonetheless, hyposmotic stress-induced changes in Vm and aKiwere blocked partly by Ba2+ (2 m m). We conclude that (i) hepatocyte Vm increases and aKidecreases during hypotonic shock; (ii) the changes in hepatocyte Vm and aKiduring and after hypotonic shock are independent of the Na+-K+ pump; (iii) the decrease in aKiduring hypotonic stress results principally from hepatocyte swelling.

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Macromolecular complexes of anionic-nonionic block copolymers and cationic antibiotic aminoglycosides have been formed by electrostatic condensation. Amphiphilicity of the complexes was introduced into the shells by incorporating a hydrophobic poly(propylene oxide) segment into the block copolymer. The resulting particles have an average hydrodynamic diameter of ~ 200 nm and contain up to 30-40 % of the drug payload. In vitro efficacies of such nanostructures in reduction of intracellular pathogens like Salmonella, Listeria, and Brucella were demonstrated. Current effort focuses on translation of this nano-drug delivery concept to in vivo model of intracellular infectious diseases. Atom transfer radical polymerization (ATRP) was utilized to prepare well-defined polymeric dispersion stabilizers that readily adsorb onto metal oxide surfaces. Two unimolecular bis(phosphonate) ATRP initiators were designed and prepared in good yield. These special initiators were successfully used to initiate polymerization of poly(N-isopropylacrylamide) (PNIPAM) in a controlled manner yielding PNIPAM with a bis(phosphonate) moiety at one terminus. The polymers readily adsorbed onto magnetite nanoparticle surfaces, thus creating thermosensitive magnetic nanostructures that form nanosized clusters upon heating above the lower critical solution temperature of PNIPAM. It is envisioned that modularity of this approach, relying on the applicability of ATRP to polymerize a vast array of monomers, could be used to prepare a library of polymeric shells for magnetic iron oxide nanoparticles. Medical intervention in drug delivery that includes detectability of drug carriers is greatly desirable. A real-time assessment of disease prognosis could be highly beneficial for developing personalized treatment strategies. As an example of this conceptual innovation, block ionomer functionalized magnetite complexes were synthesized and investigated as carriers for delivery of aminoglycosides into phagocytic cells for treatment of intracellular bacterial infections. The ionic block of copolymer contains multiple carboxylates for binding onto the iron oxide surface. The remaining unbound carboxylate anions were used to complex with cationic gentamicin in nanoshells of these complexes. The iron oxide particle core provides an imaging modality and serves as a pseudo-crosslinking site to enhance stabilities of the polyelectrolyte complexes, thus preventing them from disintegrating in the physiological environment. Currently, these hybrid complexes are being investigated in possible pharmaceutical formulations to eradicate intracellular pathogens in animal models. / Ph. D.

Mathematical modelling of cellular osmotic responses to low temperatures is being increasingly used to overcome obstacles in the successful cryopreservation of cells and tissues. Current cryobiological models often contain simplifying assumptions regarding the solution behaviour of the complicated, multisolute intra- and extra-cellular solutions. In order to obtain more accurate predictions of cryobiological outcomes, equations derived from thermodynamic principles that more accurately describe the biological solution behaviour could be used to greatly advance the design of novel cryopreservation protocols. The general hypothesis of this thesis is that the application of the multisolute osmotic virial equation, with mixing rules derived from thermodynamic first principles, to solutions of interest in cryobiology will result in more accurate predictions of the multisolute solution behaviour, which will lead to improved cryobiological modelling and increased understanding of cellular responses to cryopreservation. Specifically, this thesis demonstrates that the osmotic virial coefficients, obtained from single-solute solution data, can be used in the multisolute osmotic virial equation to accurately predict the multisolute solution behaviour, without the need to fit multisolute solution data. The form of the multisolute osmotic virial equation proposed in this thesis was used to predict the solution behaviour of a range of multisolute solutions of interest in cryobiology. The equation commonly used in cryobiology to describe cellular osmotic equilibrium is based on ideal, dilute solution assumptions. In this thesis, a non-ideal osmotic equilibrium equation was derived and, combined with the multisolute osmotic virial equation, used to more accurately predict the osmotic equilibrium of human erythrocytes. The improved equations proposed in this thesis were combined with experimental measurements of the incidence of intracellular ice formation in order to further the understanding of the role of several important cryobiological parameters on the formation of intracellular ice. This thesis work has significantly contributed to the field of cryobiology by substantially improving the accuracy of two key equations used in the modelling of cellular osmotic responses to cryopreservation. The combination of accurate mathematical modelling and results from experiments will allow increased understanding of cellular responses to cryopreservation, leading to the design of novel cryopreservation protocols. / Chemical Engineering and Medical Sciences