Five points are discussed regarding the vesicular structure isolated by fractionation techniques from the brain and liver of the guinea pig . 1 . One type of vesicle, fixed by OSO4 and shown in thin sections, is identified with the coated vesicle that has been observed in many varieties of tissues . 2. The vesicle contained in a spherical polygonal "basketwork" shown by the negative-staining techniques is identical with the coated vesicle shown in sections. 3 . Despite minute observation of this basketwork we could not confirm the existence of "hairlike projections" extending from the convex cytoplasmic surface of the vesicle . We are inclined to believe, therefore, that the hairlike projections are actually the superimposed visual images of the regular hexagons and pentagons of the network composing the basketwork . 4 . We repeat the hypothesis originally advanced by Roth and Porter (1) that the "coating" of the coated vesicle plays a role in the mechanism of the infolding and fission of the membrane ; we suggest that these events are caused by the transformation of the regular hexagons (of the coating) into regular pentagons . 5 . Finally, we make a suggestion as to the nature of those vesicles which have on their surface subparticles which look like "elementary particles (2) ." 202

The four kinds of oligopeptides specific in amino acid sequence to a rat dopamine transporter (DAT), peptide-1-peptide-4, were chemically synthethized. An attempt to produce antipeptide antibodies against these oligopeptides was made with an in vitro immunization method. Two monoclonal antibodies, MAbs H-1a and H-1b, were produced against one of the oligopeptides, peptide-1. Western blot analysis confirmed that the two antibodies recognized an approximately 85,000 Da protein in a synaptosomal fraction prepared from the rat striatum but none in the fraction from the cerebellum. The specificity of the antibody to DAT was also confirmed by an antibody absorption test using two synthetic oligopeptides, one of which is specific only to DAT. These results have confirmed the specificity of the present antibody to DAT. The expression and subcellular localization of DAT were immunohistochemically examined with MAbs H-1a and H-1b in PC12 cells treated with nerve growth factor (NGF). The antibody labeled the surface of PC12 cells. When the cells were treated with NGF, the expression of DAT was significantly emphasized, first in the area mainly including the Golgi apparatus and rough endoplasmic reticulum and then on the surface of growth cones from the beginning of neurite outgrowth. DAT was detected by Western blot analysis in a microsomal fraction prepared from PC12 cells. The activity of DAT in the PC12 cells was pharmacologically confirmed by the uptake of [3H]-dopamine and blockade by uptake inhibitors. The NGF treatment doubled the dopamine uptake activity. GBR12909, a specific inhibitor of DAT, blocked the [3H]-dopamine at a concentration of 10(-7) M. The expression of DAT and norepinephrine transporter (NET) mRNA in the PC12 cells was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). DAT mRNA significantly increased in the NGF-treated cells after 7 days of incubation, whereas NET mRNA markedly decreased.

The present study examined whether the development in rats of behavioral sensitization to methamphetamine (MAP) is related to the development of neurotoxic morphological changes presumably induced in the medial prefrontal cortex (MFC). Male rats were intraperitonieally injected with MAP (5 mg/kg) once a day for 12 days (day 1-day 12), and then the drug was withdrawn for 7-42 days (WD7-WD42). The MAP- treatment caused hypersensitivity of a successive head-movement stereotypy, which reached a basic plateau level on day 4, and rose successively to a higher level by day 12. Morphological changes were histochemically and morphometrically examined in the MFC. In the strata covering layers II and III, the densities of tyrosine hydroxylase (TH)-immunoreactive axons decreased on a daily basis to 50% of the control on day 4 and then to 40% on days 6 and 12. The densities of dopamine-,beta-hydroxylase (DBH)-immunoreactive axons did not change during the injection period. A few TUNEL-positive cells were observed in a unit area (0.25 mm2) covering layers II-V on day 6 and they increased to 19 and 16 on day 12 and WD7, respectively. These observations demonstrate a role for the neurotoxic changes in the MFC in the processes of behavioral sensitization of a stereotypy to a low dose of MAP.

Methamphetamine (MA) abuse induces deficits in cognitive performance that are related to dysfunction of the prefrontal cortex (PFC). The medial portion of the prefrontal cortex (mPFC) in rats that is crucial for cognitive function has been shown to undergo long-term potentiation (LTP) in the projections from the hippocampus. However, no study has been performed to evaluate the influence of MA on synaptic plasticity in the hippocampal-mPFC pathways. In the present experiments, we investigated the effects of repeated MA administration on hippocampal-mPFC LTP, together with MA-induced stereotyped behaviors. Repeated MA administration produced behavioral sensitization and LTP impairment in the hippocampal-mPFC pathways. The MA-induced impairment of hippocampal-mPFC LTP was prevented by the pretreatment of dopamine 1 (D1) but not dopamine 2 (D2) receptor antagonists, while D1 and D2 receptor antagonists attenuated the MA-induced stereotyped behaviors. These findings suggest that D1 receptors are crucial for the MA-induced deterioration of synaptic plasticity in the hippocampal-mPFC circuits. Impairment of LTP associated with D1 receptor dysfunction may underlie cognitive deficits in MA-dependent subjects.

Two vesicular fractions and one nonvesicular fraction were prepared from crude synaptosomes by differential centrifugation and salting out with ammonium sulfate . Fraction