b890d4e6a9a98317cb8e9c909207c04d brainsci-15-00776.pdf df1747eb53ca41954cf0adfd9b9903bb7196797e brainsci-15-00776.pdf 2f614f2c733fecdaa35bb81db89c0d359eb7a66549f5094b6ea6ecfe2bb3f06a brainsci-15-00776.pdf Title: Neonatal Handling Positively Modulates Anxiety, Sensorimotor Gating, Working Memory, and Cortico-Hippocampal Neuroplastic Adaptations in Two Genetically Selected Rat Strains Differing in Emotional and Cognitive Traits Subject: Background/Objectives: The bidirectional selection of the Roman low- (RLA) and Roman high-avoidance (RHA) rat strains for extremely slow vs. very rapid acquisition of the two-way (shuttle-box) avoidance response has generated two divergent phenotypic profiles: RHA rats exhibit a behavioural pattern and gene expression profile in the frontal cortex and hippocampus (HPC) that are relevant to social and attentional/cognitive schizophrenia-linked symptoms; on the other hand, RLA rats display phenotypic traits linked to increased anxiety and sensitivity to stress-induced depression-like behaviours. The present studies aimed to evaluate the enduring and potentially positive effects of neonatal handling-stimulation (NH) on the traits differentiating these two strains of rats. Methods: We evaluated the effects of NH on anxious behaviour, prepulse inhibition of startle (PPI), spatial working memory, and hormone responses to stress in adult rats of both strains. Furthermore, given the proposed involvement of neuronal/synaptic plasticity and neurotrophic factors in the development of anxiety, stress, depression, and schizophrenia-related symptoms, using Western blot (WB) we assessed the effects of NH on the content of brain-derived neurotrophic factor (BDNF), its trkB receptor and Polysialilated-Neural Cell Adhesion Molecule (PSA-NCAM), in the prefrontal cortex (PFC), anterior cingulate cortex (ACg), ventral (vHPC), and dorsal (dHPC) hippocampus of adult rats from both strains. Results: NH increased novelty-induced exploration and reduced anxiety, particularly in RLA rats, attenuated the stress-induced increment in corticosterone and prolactin plasma levels, and improved PPI and spatial working memory in RHA rats. These effects correlated to long-lasting increases of BDNF and PSA-NCAM content in PFC, ACg, and vHPC. Conclusions: Collectively, these findings show enduring and distinct NH effects on neuroendocrine and behavioural and cognitive processes in both rat strains, which may be linked to neuroplastic and synaptic changes in the frontal cortex and/or hippocampus. Keywords: anxiety; stress; prepulse inhibition; spatial working memory; Roman high- and low-avoidance rats; neonatal handling; BDNF; trkB; PSA-NCAM; prefrontal cortex; hippocampus Author: Cristóbal Río-Álamos, Maria P. Serra, Francesco Sanna, Maria A. Piludu, Marianna Boi, Toni Cañete, Daniel Sampedro-Viana, Ignasi Oliveras, Adolf Tobeña, Maria G. Corda, Osvaldo Giorgi, Alberto Fernández-Teruel and Marina Quartu Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Tue Jul 22 05:47:39 2025 CEST ModDate: Tue Jul 22 05:50:52 2025 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 24 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1472631 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- FZPVKN+URWPalladioL-Bold Type 1 Custom yes yes yes 147 0 KQDOAA+URWPalladioL-Roma Type 1 Custom yes yes yes 148 0 MLKADY+URWPalladioL-Ital Type 1 Custom yes yes yes 149 0 XEYJZU+CMSY10 Type 1 Builtin yes yes yes 313 0 GMIAEB+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 305 0 GMIADA+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 306 0 GMIADB+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 307 0 GMIBLO+TimesNewRomanPSMT TrueType WinAnsi yes yes no 308 0 AZDANO+CMBSY10 Type 1 Builtin yes yes yes 357 0 LOEGIB+PalatinoLinotype TrueType WinAnsi yes yes no 370 0 LOEGHA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 371 0 OVNEXW+EURM10 Type 1 Builtin yes yes yes 400 0 LOEGIB+PalatinoLinotype TrueType WinAnsi yes yes no 445 0 LOEINI+TimesNewRoman TrueType WinAnsi yes yes no 446 0 LOEGIB+PalatinoLinotype TrueType WinAnsi yes yes no 452 0 LOEINI+TimesNewRoman TrueType WinAnsi yes yes no 453 0 LOEGIB+PalatinoLinotype TrueType WinAnsi yes yes no 481 0 LOEINI+TimesNewRoman TrueType WinAnsi yes yes no 482 0 LOEGIB+PalatinoLinotype TrueType WinAnsi yes yes no 509 0 LOEINI+TimesNewRoman TrueType WinAnsi yes yes no 510 0 YKYMMN+VnURWPalladioL Type 1 Custom yes yes yes 711 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-09-10 03:08:09 CEST RepresentationInformation: brainsci-15-00776.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-09-09 12:13:30 CEST Size: 1472631 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 892 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Material and Methods Children: Item: Title: Animals and Neonatal Handling (NH) Treatment Item: Title: Behavioural and Hormonal Measures Children: Item: Title: Novel Object Exploration (NOE) Test of Anxiety-Related Behaviour Item: Title: Elevated Zero-Maze (EZM) Test of Anxiety Item: Title: Baseline Startle and Prepulse Inhibition of the Acoustic Startle Response (Sensorimotor Gating, PPI) Item: Title: Morris Water Maze: Delayed Matching-to-Place Task (DMTP) of Spatial Working Memory Item: Title: Hormone Measurements Item: Title: Brain Sampling and Molecular Measures Item: Title: Statistical Analyses Item: Title: Results Children: Item: Title: Novel Object Exploration (NOE) and Elevated Zero-Maze (EZM) Tests Item: Title: Baseline Startle Response and Prepulse Inhibition of the Startle Response (PPI) Item: Title: Delayed Matching-to-Place (DMPT) Task of Spatial Working Memory Item: Title: Baseline and Post-Stress Corticosterone and Prolactin Responses Item: Title: BDNF, trkB, and PSA-NCAM Protein Levels in PFC (Prelimbic/Infralimbic) and ACg of RHA and RLA Rats: Effects of Neonatal Handling Item: Title: BDNF, trkB, and PSA-NCAM Protein Levels in vHPC and dHPC of RHA and RLA Rats: Effects of Neonatal Handling Item: Title: Correlational Analyses: Relations Among BDNF, trkB, and PSA-NCAM Protein Levels in the Different Areas, and Between Specific Protein Levels and Behaviour, Hormonal, and Cognitive Measures Item: Title: Exploratory Factor Analysis of Protein Levels, Behaviour, and Stress Hormone Values Item: Title: Discussion Item: Title: Conclusions Item: Title: References Info: Title: Neonatal Handling Positively Modulates Anxiety, Sensorimotor Gating, Working Memory, and Cortico-Hippocampal Neuroplastic Adaptations in Two Genetically Selected Rat Strains Differing in Emotional and Cognitive Traits Author: Cristóbal Río-Álamos, Maria P. Serra, Francesco Sanna, Maria A. Piludu, Marianna Boi, Toni Cañete, Daniel Sampedro-Viana, Ignasi Oliveras, Adolf Tobeña, Maria G. Corda, Osvaldo Giorgi, Alberto Fernández-Teruel and Marina Quartu Subject: Background/Objectives: The bidirectional selection of the Roman low- (RLA) and Roman high-avoidance (RHA) rat strains for extremely slow vs. very rapid acquisition of the two-way (shuttle-box) avoidance response has generated two divergent phenotypic profiles: RHA rats exhibit a behavioural pattern and gene expression profile in the frontal cortex and hippocampus (HPC) that are relevant to social and attentional/cognitive schizophrenia-linked symptoms; on the other hand, RLA rats display phenotypic traits linked to increased anxiety and sensitivity to stress-induced depression-like behaviours. The present studies aimed to evaluate the enduring and potentially positive effects of neonatal handling-stimulation (NH) on the traits differentiating these two strains of rats. Methods: We evaluated the effects of NH on anxious behaviour, prepulse inhibition of startle (PPI), spatial working memory, and hormone responses to stress in adult rats of both strains. Furthermore, given the proposed involvement of neuronal/synaptic plasticity and neurotrophic factors in the development of anxiety, stress, depression, and schizophrenia-related symptoms, using Western blot (WB) we assessed the effects of NH on the content of brain-derived neurotrophic factor (BDNF), its trkB receptor and Polysialilated-Neural Cell Adhesion Molecule (PSA-NCAM), in the prefrontal cortex (PFC), anterior cingulate cortex (ACg), ventral (vHPC), and dorsal (dHPC) hippocampus of adult rats from both strains. Results: NH increased novelty-induced exploration and reduced anxiety, particularly in RLA rats, attenuated the stress-induced increment in corticosterone and prolactin plasma levels, and improved PPI and spatial working memory in RHA rats. These effects correlated to long-lasting increases of BDNF and PSA-NCAM content in PFC, ACg, and vHPC. Conclusions: Collectively, these findings show enduring and distinct NH effects on neuroendocrine and behavioural and cognitive processes in both rat strains, which may be linked to neuroplastic and synaptic changes in the frontal cortex and/or hippocampus. Keywords: anxiety; stress; prepulse inhibition; spatial working memory; Roman high- and low-avoidance rats; neonatal handling; BDNF; trkB; PSA-NCAM; prefrontal cortex; hippocampus Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Tue Jul 22 05:47:39 CEST 2025 ModDate: Tue Jul 22 05:50:52 CEST 2025 ID: 0x8ab5f77c78e8c59cf5298b1c524daf86, 0x46250928063571311271b814a46c8faf Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: OVNEXW+EURM10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: OVNEXW+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Font: BaseFont: FZPVKN+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 243 FontDescriptor: FontName: FZPVKN+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: KQDOAA+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: KQDOAA+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: MLKADY+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 243 FontDescriptor: FontName: MLKADY+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: AZDANO+CMBSY10 FontSubset: true FirstChar: 0 LastChar: 6 FontDescriptor: FontName: AZDANO+CMBSY10 Flags: Symbolic FontBBox: -26, -940, 1332, 825 FontFile: true ToUnicode: true Font: BaseFont: YKYMMN+VnURWPalladioL FontSubset: true FirstChar: 177 LastChar: 254 FontDescriptor: FontName: YKYMMN+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: XEYJZU+CMSY10 FontSubset: true FirstChar: 0 LastChar: 20 FontDescriptor: FontName: XEYJZU+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 89, 177, 152, 186 Annotation: Subtype: Link Rect: 34, 166, 115, 174 Annotation: Subtype: Link Rect: 36, 75, 152, 84 Annotation: Subtype: Link Rect: 34, 63, 89, 72 Annotation: Subtype: Link Rect: 34, 774, 179, 810 Annotation: Subtype: Link Rect: 515, 774, 560, 810 Annotation: Subtype: Link Rect: 221, 623, 232, 636 Annotation: Subtype: Link Rect: 317, 623, 328, 636 Annotation: Subtype: Link Rect: 485, 623, 496, 636 Annotation: Subtype: Link Rect: 95, 608, 106, 622 Annotation: Subtype: Link Rect: 226, 608, 237, 622 Annotation: Subtype: Link Rect: 323, 608, 334, 622 Annotation: Subtype: Link Rect: 406, 608, 417, 622 Annotation: Subtype: Link Rect: 112, 595, 123, 608 Annotation: Subtype: Link Rect: 360, 595, 371, 608 Annotation: Subtype: Link Rect: 408, 27, 560, 37 Annotation: Subtype: Link Rect: 34, 391, 87, 410 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 361, 633, 368, 642 ActionDest: 111 Annotation: Subtype: Link Rect: 371, 633, 377, 642 ActionDest: 111 Annotation: Subtype: Link Rect: 445, 577, 452, 586 ActionDest: 111 Annotation: Subtype: Link Rect: 455, 577, 462, 586 ActionDest: 111 Annotation: Subtype: Link Rect: 471, 494, 478, 503 ActionDest: 111 Annotation: Subtype: Link Rect: 481, 494, 488, 503 ActionDest: 111 Annotation: Subtype: Link Rect: 488, 494, 500, 503 ActionDest: 111 Annotation: Subtype: Link Rect: 503, 494, 515, 503 ActionDest: 111 Annotation: Subtype: Link Rect: 401, 480, 408, 489 ActionDest: 111 Annotation: Subtype: Link Rect: 490, 396, 497, 405 ActionDest: 111 Annotation: Subtype: Link Rect: 500, 396, 507, 405 ActionDest: 111 Annotation: Subtype: Link Rect: 507, 396, 519, 405 ActionDest: 223 Annotation: Subtype: Link Rect: 522, 396, 534, 405 ActionDest: 223 Annotation: Subtype: Link Rect: 288, 341, 295, 350 ActionDest: 111 Annotation: Subtype: Link Rect: 520, 327, 527, 336 ActionDest: 111 Annotation: Subtype: Link Rect: 528, 327, 540, 336 ActionDest: 223 Annotation: Subtype: Link Rect: 540, 327, 552, 336 ActionDest: 223 Annotation: Subtype: Link Rect: 518, 313, 525, 322 ActionDest: 111 Annotation: Subtype: Link Rect: 307, 285, 314, 294 ActionDest: 111 Annotation: Subtype: Link Rect: 314, 285, 321, 294 ActionDest: 111 Annotation: Subtype: Link Rect: 320, 243, 327, 252 ActionDest: 111 Annotation: Subtype: Link Rect: 330, 243, 337, 252 ActionDest: 111 Annotation: Subtype: Link Rect: 338, 243, 350, 252 ActionDest: 223 Annotation: Subtype: Link Rect: 353, 243, 364, 252 ActionDest: 223 Annotation: Subtype: Link Rect: 294, 215, 301, 224 ActionDest: 111 Annotation: Subtype: Link Rect: 302, 215, 314, 224 ActionDest: 223 Annotation: Subtype: Link Rect: 192, 201, 199, 210 ActionDest: 111 Annotation: Subtype: Link Rect: 199, 201, 206, 210 ActionDest: 111 Annotation: Subtype: Link Rect: 207, 201, 219, 210 ActionDest: 223 Annotation: Subtype: Link Rect: 219, 201, 231, 210 ActionDest: 223 Annotation: Subtype: Link Rect: 232, 201, 244, 210 ActionDest: 223 Annotation: Subtype: Link Rect: 247, 187, 254, 196 ActionDest: 111 Annotation: Subtype: Link Rect: 257, 187, 264, 196 ActionDest: 111 Annotation: Subtype: Link Rect: 265, 187, 272, 196 ActionDest: 111 Annotation: Subtype: Link Rect: 272, 187, 279, 196 ActionDest: 111 Annotation: Subtype: Link Rect: 280, 187, 292, 196 ActionDest: 223 Annotation: Subtype: Link Rect: 292, 187, 304, 196 ActionDest: 223 Annotation: Subtype: Link Rect: 230, 159, 237, 168 ActionDest: 111 Annotation: Subtype: Link Rect: 237, 160, 244, 168 ActionDest: 111 Annotation: Subtype: Link Rect: 319, 160, 326, 168 ActionDest: 111 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 449, 745, 456, 754 ActionDest: 111 Annotation: Subtype: Link Rect: 459, 745, 466, 754 ActionDest: 111 Annotation: Subtype: Link Rect: 466, 744, 473, 754 ActionDest: 111 Annotation: Subtype: Link Rect: 474, 745, 486, 754 ActionDest: 223 Annotation: Subtype: Link Rect: 220, 661, 232, 670 ActionDest: 223 Annotation: Subtype: Link Rect: 235, 661, 247, 670 ActionDest: 223 Annotation: Subtype: Link Rect: 192, 619, 204, 628 ActionDest: 223 Annotation: Subtype: Link Rect: 205, 619, 217, 628 ActionDest: 223 Annotation: Subtype: Link Rect: 374, 605, 386, 614 ActionDest: 223 Annotation: Subtype: Link Rect: 483, 591, 495, 600 ActionDest: 223 Annotation: Subtype: Link Rect: 496, 591, 508, 600 ActionDest: 223 Annotation: Subtype: Link Rect: 508, 591, 520, 600 ActionDest: 223 Annotation: Subtype: Link Rect: 398, 577, 410, 586 ActionDest: 223 Annotation: Subtype: Link Rect: 360, 522, 367, 531 ActionDest: 111 Annotation: Subtype: Link Rect: 367, 522, 379, 531 ActionDest: 223 Annotation: Subtype: Link Rect: 515, 494, 527, 503 ActionDest: 223 Annotation: Subtype: Link Rect: 353, 466, 365, 475 ActionDest: 223 Annotation: Subtype: Link Rect: 449, 410, 461, 419 ActionDest: 223 Annotation: Subtype: Link Rect: 461, 410, 473, 419 ActionDest: 223 Annotation: Subtype: Link Rect: 210, 382, 222, 391 ActionDest: 223 Annotation: Subtype: Link Rect: 222, 382, 234, 391 ActionDest: 223 Annotation: Subtype: Link Rect: 481, 354, 493, 364 ActionDest: 223 Annotation: Subtype: Link Rect: 279, 327, 291, 336 ActionDest: 223 Annotation: Subtype: Link Rect: 291, 327, 303, 336 ActionDest: 287 Annotation: Subtype: Link Rect: 306, 327, 318, 336 ActionDest: 287 Annotation: Subtype: Link Rect: 419, 313, 431, 322 ActionDest: 287 Annotation: Subtype: Link Rect: 432, 313, 444, 322 ActionDest: 287 Annotation: Subtype: Link Rect: 531, 285, 543, 294 ActionDest: 287 Annotation: Subtype: Link Rect: 544, 285, 556, 294 ActionDest: 287 Annotation: Subtype: Link Rect: 352, 243, 364, 252 ActionDest: 287 Annotation: Subtype: Link Rect: 209, 215, 215, 224 ActionDest: 111 Annotation: Subtype: Link Rect: 216, 215, 228, 224 ActionDest: 223 Annotation: Subtype: Link Rect: 291, 201, 298, 210 ActionDest: 111 Annotation: Subtype: Link Rect: 525, 160, 537, 169 ActionDest: 287 Annotation: Subtype: Link Rect: 540, 159, 552, 169 ActionDest: 287 Annotation: Subtype: Link Rect: 431, 118, 443, 127 ActionDest: 287 Annotation: Subtype: Link Rect: 443, 118, 455, 127 ActionDest: 287 Annotation: Subtype: Link Rect: 458, 118, 470, 127 ActionDest: 287 Annotation: Subtype: Link Rect: 471, 104, 483, 113 ActionDest: 111 Annotation: Subtype: Link Rect: 484, 104, 496, 113 ActionDest: 223 Annotation: Subtype: Link Rect: 496, 104, 508, 113 ActionDest: 287 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 293, 571, 300, 583 ActionDest: 15 Annotation: Subtype: Link Rect: 274, 518, 286, 527 ActionDest: 111 Annotation: Subtype: Link Rect: 287, 518, 299, 527 ActionDest: 223 Annotation: Subtype: Link Rect: 358, 376, 365, 388 ActionDest: 15 Annotation: Subtype: Link Rect: 456, 179, 463, 192 ActionDest: 15 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 459, 673, 466, 685 ActionDest: 15 Annotation: Subtype: Link Rect: 273, 606, 285, 615 ActionDest: 111 Annotation: Subtype: Link Rect: 285, 606, 297, 615 ActionDest: 223 Annotation: Subtype: Link Rect: 534, 437, 541, 449 ActionDest: 60 Annotation: Subtype: Link Rect: 191, 423, 198, 435 ActionDest: 163 Annotation: Subtype: Link Rect: 477, 410, 484, 422 ActionDest: 15 Annotation: Subtype: Link Rect: 262, 259, 274, 268 ActionDest: 223 Annotation: Subtype: Link Rect: 487, 204, 494, 216 ActionDest: 15 Annotation: Subtype: Link Rect: 321, 193, 333, 202 ActionDest: 111 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 186, 503, 192, 511 ActionDest: 111 Annotation: Subtype: Link Rect: 192, 503, 203, 511 ActionDest: 111 Annotation: Subtype: Link Rect: 298, 465, 309, 473 ActionDest: 111 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 246, 571, 258, 580 ActionDest: 223 Annotation: Subtype: Link Rect: 486, 558, 498, 566 ActionDest: 111 Annotation: Subtype: Link Rect: 453, 438, 460, 450 ActionDest: 15 Annotation: Subtype: Link Rect: 533, 396, 540, 408 ActionDest: 45 Annotation: Subtype: Link Rect: 346, 66, 353, 78 ActionDest: 45 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 329, 717, 341, 726 ActionDest: 111 Annotation: Subtype: Link Rect: 339, 689, 351, 698 ActionDest: 111 Annotation: Subtype: Link Rect: 326, 618, 333, 630 ActionDest: 15 Annotation: Subtype: Link Rect: 199, 313, 206, 325 ActionDest: 15 Annotation: Subtype: Link Rect: 319, 273, 331, 283 ActionDest: 287 Annotation: Subtype: Link Rect: 456, 260, 468, 269 ActionDest: 287 Annotation: Subtype: Link Rect: 518, 163, 530, 172 ActionDest: 287 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 376, 421, 559, 434 Annotation: Subtype: Link Rect: 286, 258, 298, 267 ActionDest: 287 Annotation: Subtype: Link Rect: 299, 258, 311, 267 ActionDest: 287 Annotation: Subtype: Link Rect: 314, 258, 326, 267 ActionDest: 418 Annotation: Subtype: Link Rect: 323, 189, 335, 198 ActionDest: 418 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 332, 544, 339, 555 ActionDest: 163 Annotation: Subtype: Link Rect: 549, 488, 556, 500 ActionDest: 163 Annotation: Subtype: Link Rect: 382, 432, 389, 444 ActionDest: 163 Annotation: Subtype: Link Rect: 346, 350, 353, 362 ActionDest: 163 Annotation: Subtype: Link Rect: 399, 336, 406, 348 ActionDest: 163 Annotation: Subtype: Link Rect: 250, 294, 257, 306 ActionDest: 163 Annotation: Subtype: Link Rect: 506, 294, 513, 306 ActionDest: 163 Annotation: Subtype: Link Rect: 398, 267, 405, 278 ActionDest: 163 Annotation: Subtype: Link Rect: 316, 198, 323, 210 ActionDest: 163 Annotation: Subtype: Link Rect: 258, 170, 265, 182 ActionDest: 163 Annotation: Subtype: Link Rect: 551, 156, 558, 168 ActionDest: 163 Annotation: Subtype: Link Rect: 457, 74, 464, 86 ActionDest: 163 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 303, 743, 310, 754 ActionDest: 163 Annotation: Subtype: Link Rect: 549, 714, 556, 726 ActionDest: 163 Annotation: Subtype: Link Rect: 311, 659, 318, 670 ActionDest: 163 Annotation: Subtype: Link Rect: 217, 578, 224, 590 ActionDest: 83 Annotation: Subtype: Link Rect: 383, 564, 390, 576 ActionDest: 83 Annotation: Subtype: Link Rect: 196, 550, 203, 562 ActionDest: 88 Annotation: Subtype: Link Rect: 207, 509, 214, 521 ActionDest: 88 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 302, 236, 309, 248 ActionDest: 83 Annotation: Subtype: Link Rect: 380, 236, 387, 248 ActionDest: 88 Annotation: Subtype: Link Rect: 244, 208, 251, 220 ActionDest: 83 Annotation: Subtype: Link Rect: 296, 194, 303, 206 ActionDest: 88 Annotation: Subtype: Link Rect: 476, 180, 483, 192 ActionDest: 83 Annotation: Subtype: Link Rect: 508, 180, 515, 192 ActionDest: 88 Annotation: Subtype: Link Rect: 298, 114, 305, 126 ActionDest: 499 Annotation: Subtype: Link Rect: 242, 100, 249, 112 ActionDest: 499 Annotation: Subtype: Link Rect: 403, 86, 410, 98 ActionDest: 499 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 196, 201, 203, 213 ActionDest: 499 Annotation: Subtype: Link Rect: 502, 201, 509, 213 ActionDest: 499 Annotation: Subtype: Link Rect: 311, 173, 318, 185 ActionDest: 93 Annotation: Subtype: Link Rect: 241, 145, 248, 157 ActionDest: 93 Annotation: Subtype: Link Rect: 368, 117, 375, 129 ActionDest: 93 Annotation: Subtype: Link Rect: 218, 89, 225, 101 ActionDest: 93 Page: Sequence: 14 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 435, 520, 442, 532 ActionDest: 101 Annotation: Subtype: Link Rect: 541, 465, 548, 477 ActionDest: 101 Annotation: Subtype: Link Rect: 251, 353, 258, 365 ActionDest: 101 Annotation: Subtype: Link Rect: 265, 311, 272, 323 ActionDest: 101 Annotation: Subtype: Link Rect: 194, 173, 201, 184 ActionDest: 101 Page: Sequence: 16 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 387, 689, 394, 698 ActionDest: 111 Annotation: Subtype: Link Rect: 395, 689, 401, 698 ActionDest: 111 Annotation: Subtype: Link Rect: 402, 689, 413, 698 ActionDest: 111 Annotation: Subtype: Link Rect: 414, 689, 425, 698 ActionDest: 111 Annotation: Subtype: Link Rect: 426, 689, 437, 698 ActionDest: 418 Annotation: Subtype: Link Rect: 510, 689, 517, 698 ActionDest: 111 Annotation: Subtype: Link Rect: 518, 689, 524, 698 ActionDest: 111 Annotation: Subtype: Link Rect: 525, 689, 532, 698 ActionDest: 111 Annotation: Subtype: Link Rect: 532, 689, 544, 698 ActionDest: 223 Annotation: Subtype: Link Rect: 544, 689, 556, 698 ActionDest: 223 Annotation: Subtype: Link Rect: 382, 647, 394, 656 ActionDest: 111 Annotation: Subtype: Link Rect: 328, 605, 340, 614 ActionDest: 111 Annotation: Subtype: Link Rect: 340, 605, 352, 614 ActionDest: 287 Annotation: Subtype: Link Rect: 190, 591, 202, 600 ActionDest: 287 Annotation: Subtype: Link Rect: 205, 591, 217, 600 ActionDest: 287 Annotation: Subtype: Link Rect: 217, 591, 229, 600 ActionDest: 287 Annotation: Subtype: Link Rect: 231, 591, 243, 600 ActionDest: 418 Annotation: Subtype: Link Rect: 243, 591, 255, 600 ActionDest: 418 Annotation: Subtype: Link Rect: 403, 536, 415, 545 ActionDest: 287 Annotation: Subtype: Link Rect: 416, 536, 428, 545 ActionDest: 287 Annotation: Subtype: Link Rect: 428, 536, 440, 545 ActionDest: 418 Annotation: Subtype: Link Rect: 182, 522, 194, 531 ActionDest: 418 Annotation: Subtype: Link Rect: 324, 438, 331, 447 ActionDest: 111 Annotation: Subtype: Link Rect: 334, 438, 341, 447 ActionDest: 111 Annotation: Subtype: Link Rect: 342, 438, 349, 447 ActionDest: 111 Annotation: Subtype: Link Rect: 352, 438, 364, 447 ActionDest: 223 Annotation: Subtype: Link Rect: 364, 438, 376, 447 ActionDest: 223 Annotation: Subtype: Link Rect: 379, 438, 391, 447 ActionDest: 223 Annotation: Subtype: Link Rect: 254, 327, 261, 336 ActionDest: 111 Annotation: Subtype: Link Rect: 261, 327, 273, 336 ActionDest: 111 Annotation: Subtype: Link Rect: 274, 327, 286, 336 ActionDest: 223 Annotation: Subtype: Link Rect: 287, 327, 298, 336 ActionDest: 223 Annotation: Subtype: Link Rect: 299, 327, 311, 336 ActionDest: 418 Annotation: Subtype: Link Rect: 285, 271, 291, 280 ActionDest: 111 Annotation: Subtype: Link Rect: 292, 271, 304, 280 ActionDest: 418 Annotation: Subtype: Link Rect: 307, 271, 319, 280 ActionDest: 418 Annotation: Subtype: Link Rect: 196, 229, 208, 238 ActionDest: 418 Annotation: Subtype: Link Rect: 263, 173, 275, 182 ActionDest: 418 Annotation: Subtype: Link Rect: 275, 173, 287, 182 ActionDest: 418 Annotation: Subtype: Link Rect: 271, 76, 278, 85 ActionDest: 111 Annotation: Subtype: Link Rect: 281, 76, 288, 85 ActionDest: 111 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 492, 731, 499, 740 ActionDest: 111 Annotation: Subtype: Link Rect: 502, 731, 509, 740 ActionDest: 111 Annotation: Subtype: Link Rect: 357, 703, 364, 712 ActionDest: 111 Annotation: Subtype: Link Rect: 367, 703, 374, 712 ActionDest: 111 Annotation: Subtype: Link Rect: 375, 703, 382, 712 ActionDest: 111 Annotation: Subtype: Link Rect: 385, 703, 397, 712 ActionDest: 223 Annotation: Subtype: Link Rect: 397, 703, 409, 712 ActionDest: 287 Annotation: Subtype: Link Rect: 410, 703, 422, 712 ActionDest: 418 Annotation: Subtype: Link Rect: 422, 703, 434, 712 ActionDest: 418 Annotation: Subtype: Link Rect: 217, 675, 224, 684 ActionDest: 111 Annotation: Subtype: Link Rect: 360, 591, 372, 600 ActionDest: 418 Annotation: Subtype: Link Rect: 528, 564, 540, 572 ActionDest: 223 Annotation: Subtype: Link Rect: 540, 563, 552, 572 ActionDest: 223 Annotation: Subtype: Link Rect: 362, 522, 374, 531 ActionDest: 223 Annotation: Subtype: Link Rect: 374, 522, 386, 531 ActionDest: 223 Annotation: Subtype: Link Rect: 349, 452, 361, 461 ActionDest: 111 Annotation: Subtype: Link Rect: 362, 452, 374, 461 ActionDest: 287 Annotation: Subtype: Link Rect: 377, 452, 388, 461 ActionDest: 287 Annotation: Subtype: Link Rect: 389, 452, 401, 461 ActionDest: 287 Annotation: Subtype: Link Rect: 404, 452, 416, 461 ActionDest: 287 Annotation: Subtype: Link Rect: 416, 452, 428, 461 ActionDest: 287 Annotation: Subtype: Link Rect: 429, 452, 441, 461 ActionDest: 418 Annotation: Subtype: Link Rect: 441, 452, 453, 461 ActionDest: 418 Annotation: Subtype: Link Rect: 456, 452, 468, 461 ActionDest: 418 Annotation: Subtype: Link Rect: 374, 285, 386, 294 ActionDest: 111 Annotation: Subtype: Link Rect: 313, 199, 320, 211 ActionDest: 101 Annotation: Subtype: Link Rect: 460, 157, 467, 169 ActionDest: 101 Annotation: Subtype: Link Rect: 272, 87, 279, 99 ActionDest: 101 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 192, 536, 204, 545 ActionDest: 418 Annotation: Subtype: Link Rect: 205, 536, 217, 545 ActionDest: 418 Annotation: Subtype: Link Rect: 286, 536, 298, 545 ActionDest: 418 Annotation: Subtype: Link Rect: 251, 508, 263, 517 ActionDest: 223 Annotation: Subtype: Link Rect: 263, 508, 275, 517 ActionDest: 223 Annotation: Subtype: Link Rect: 291, 327, 303, 336 ActionDest: 223 Annotation: Subtype: Link Rect: 303, 327, 315, 336 ActionDest: 223 Annotation: Subtype: Link Rect: 384, 327, 391, 336 ActionDest: 111 Annotation: Subtype: Link Rect: 395, 327, 401, 336 ActionDest: 111 Annotation: Subtype: Link Rect: 347, 299, 354, 308 ActionDest: 111 Annotation: Subtype: Link Rect: 357, 299, 364, 308 ActionDest: 111 Annotation: Subtype: Link Rect: 365, 299, 377, 308 ActionDest: 111 Annotation: Subtype: Link Rect: 377, 299, 389, 308 ActionDest: 418 Annotation: Subtype: Link Rect: 514, 271, 526, 280 ActionDest: 111 Annotation: Subtype: Link Rect: 288, 201, 300, 210 ActionDest: 287 Annotation: Subtype: Link Rect: 300, 201, 312, 210 ActionDest: 287 Annotation: Subtype: Link Rect: 376, 132, 387, 141 ActionDest: 111 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 525, 745, 537, 754 ActionDest: 111 Annotation: Subtype: Link Rect: 188, 731, 200, 740 ActionDest: 287 Annotation: Subtype: Link Rect: 201, 730, 213, 740 ActionDest: 287 Annotation: Subtype: Link Rect: 490, 479, 502, 488 ActionDest: 223 Annotation: Subtype: Link Rect: 503, 479, 515, 488 ActionDest: 223 Annotation: Subtype: Link Rect: 515, 479, 527, 488 ActionDest: 287 Annotation: Subtype: Link Rect: 528, 479, 540, 488 ActionDest: 287 Annotation: Subtype: Link Rect: 540, 479, 552, 488 ActionDest: 418 Annotation: Subtype: Link Rect: 487, 354, 499, 363 ActionDest: 111 Annotation: Subtype: Link Rect: 499, 353, 511, 363 ActionDest: 287 Annotation: Subtype: Link Rect: 512, 353, 524, 363 ActionDest: 287 Annotation: Subtype: Link Rect: 527, 353, 539, 363 ActionDest: 418 Annotation: Subtype: Link Rect: 539, 353, 551, 363 ActionDest: 418 Annotation: Subtype: Link Rect: 536, 326, 560, 337 Annotation: Subtype: Link Rect: 165, 312, 390, 323 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 59, 517, 96, 527 Annotation: Subtype: Link Rect: 102, 517, 138, 527 Annotation: Subtype: Link Rect: 95, 466, 133, 476 Annotation: Subtype: Link Rect: 139, 466, 175, 476 Annotation: Subtype: Link Rect: 141, 427, 179, 437 Annotation: Subtype: Link Rect: 185, 427, 221, 437 Annotation: Subtype: Link Rect: 59, 389, 96, 399 Annotation: Subtype: Link Rect: 102, 389, 138, 399 Annotation: Subtype: Link Rect: 262, 363, 300, 374 Annotation: Subtype: Link Rect: 306, 363, 342, 374 Annotation: Subtype: Link Rect: 294, 337, 332, 348 Annotation: Subtype: Link Rect: 338, 337, 374, 348 Annotation: Subtype: Link Rect: 59, 312, 96, 322 Annotation: Subtype: Link Rect: 102, 312, 138, 322 Annotation: Subtype: Link Rect: 447, 286, 485, 297 Annotation: Subtype: Link Rect: 491, 286, 527, 297 Annotation: Subtype: Link Rect: 519, 260, 557, 271 Annotation: Subtype: Link Rect: 59, 249, 95, 259 Annotation: Subtype: Link Rect: 86, 210, 124, 220 Annotation: Subtype: Link Rect: 130, 210, 166, 220 Annotation: Subtype: Link Rect: 197, 159, 235, 169 Annotation: Subtype: Link Rect: 241, 159, 277, 169 Annotation: Subtype: Link Rect: 131, 108, 168, 118 Annotation: Subtype: Link Rect: 175, 108, 211, 118 Annotation: Subtype: Link Rect: 140, 70, 177, 80 Annotation: Subtype: Link Rect: 184, 70, 220, 80 Page: Sequence: 22 Annotations: Annotation: Subtype: Link Rect: 398, 731, 436, 742 Annotation: Subtype: Link Rect: 442, 731, 478, 742 Annotation: Subtype: Link Rect: 59, 693, 96, 703 Annotation: Subtype: Link Rect: 102, 693, 138, 703 Annotation: Subtype: Link Rect: 446, 654, 484, 665 Annotation: Subtype: Link Rect: 490, 654, 526, 665 Annotation: Subtype: Link Rect: 175, 616, 212, 627 Annotation: Subtype: Link Rect: 218, 616, 254, 627 Annotation: Subtype: Link Rect: 371, 577, 408, 588 Annotation: Subtype: Link Rect: 414, 577, 450, 588 Annotation: Subtype: Link Rect: 375, 552, 413, 563 Annotation: Subtype: Link Rect: 419, 552, 455, 563 Annotation: Subtype: Link Rect: 467, 526, 505, 537 Annotation: Subtype: Link Rect: 511, 526, 547, 537 Annotation: Subtype: Link Rect: 519, 501, 557, 512 Annotation: Subtype: Link Rect: 59, 489, 95, 499 Annotation: Subtype: Link Rect: 131, 463, 168, 473 Annotation: Subtype: Link Rect: 175, 463, 211, 473 Annotation: Subtype: Link Rect: 59, 425, 96, 435 Annotation: Subtype: Link Rect: 102, 425, 138, 435 Annotation: Subtype: Link Rect: 233, 399, 270, 410 Annotation: Subtype: Link Rect: 277, 399, 313, 410 Annotation: Subtype: Link Rect: 519, 360, 557, 371 Annotation: Subtype: Link Rect: 59, 348, 95, 358 Annotation: Subtype: Link Rect: 104, 310, 142, 320 Annotation: Subtype: Link Rect: 148, 310, 184, 320 Annotation: Subtype: Link Rect: 337, 284, 375, 295 Annotation: Subtype: Link Rect: 381, 284, 417, 295 Annotation: Subtype: Link Rect: 236, 259, 274, 269 Annotation: Subtype: Link Rect: 280, 259, 316, 269 Annotation: Subtype: Link Rect: 183, 233, 220, 244 Annotation: Subtype: Link Rect: 227, 233, 263, 244 Annotation: Subtype: Link Rect: 379, 207, 417, 218 Annotation: Subtype: Link Rect: 423, 207, 459, 218 Annotation: Subtype: Link Rect: 95, 182, 133, 193 Annotation: Subtype: Link Rect: 139, 182, 175, 193 Annotation: Subtype: Link Rect: 254, 156, 291, 167 Annotation: Subtype: Link Rect: 297, 156, 333, 167 Annotation: Subtype: Link Rect: 375, 130, 413, 141 Annotation: Subtype: Link Rect: 419, 130, 455, 141 Annotation: Subtype: Link Rect: 190, 93, 227, 103 Annotation: Subtype: Link Rect: 234, 93, 270, 103 Annotation: Subtype: Link Rect: 397, 67, 435, 78 Annotation: Subtype: Link Rect: 441, 67, 477, 78 Page: Sequence: 23 Annotations: Annotation: Subtype: Link Rect: 262, 731, 299, 742 Annotation: Subtype: Link Rect: 305, 731, 341, 742 Annotation: Subtype: Link Rect: 228, 705, 264, 716 Annotation: Subtype: Link Rect: 82, 668, 119, 678 Annotation: Subtype: Link Rect: 125, 668, 161, 678 Annotation: Subtype: Link Rect: 382, 641, 419, 652 Annotation: Subtype: Link Rect: 426, 641, 462, 652 Annotation: Subtype: Link Rect: 472, 616, 510, 627 Annotation: Subtype: Link Rect: 516, 616, 552, 627 Annotation: Subtype: Link Rect: 381, 590, 419, 601 Annotation: Subtype: Link Rect: 425, 590, 461, 601 Annotation: Subtype: Link Rect: 274, 565, 312, 576 Annotation: Subtype: Link Rect: 318, 565, 354, 576 Annotation: Subtype: Link Rect: 194, 527, 231, 537 Annotation: Subtype: Link Rect: 238, 527, 274, 537 Annotation: Subtype: Link Rect: 193, 489, 230, 499 Annotation: Subtype: Link Rect: 236, 489, 272, 499 Annotation: Subtype: Link Rect: 473, 475, 510, 486 Annotation: Subtype: Link Rect: 516, 475, 552, 486 Annotation: Subtype: Link Rect: 140, 450, 177, 461 Annotation: Subtype: Link Rect: 184, 450, 220, 461 Annotation: Subtype: Link Rect: 372, 424, 410, 435 Annotation: Subtype: Link Rect: 416, 424, 452, 435 Annotation: Subtype: Link Rect: 519, 399, 557, 410 Annotation: Subtype: Link Rect: 59, 387, 95, 397 Annotation: Subtype: Link Rect: 306, 360, 344, 371 Annotation: Subtype: Link Rect: 350, 360, 386, 371 Annotation: Subtype: Link Rect: 520, 335, 557, 346 Annotation: Subtype: Link Rect: 59, 323, 95, 333 Annotation: Subtype: Link Rect: 207, 284, 244, 295 Annotation: Subtype: Link Rect: 251, 284, 287, 295 Annotation: Subtype: Link Rect: 59, 246, 96, 256 Annotation: Subtype: Link Rect: 102, 246, 138, 256 Annotation: Subtype: Link Rect: 393, 220, 431, 231 Annotation: Subtype: Link Rect: 437, 220, 473, 231 Annotation: Subtype: Link Rect: 422, 194, 460, 205 Annotation: Subtype: Link Rect: 466, 194, 502, 205 Annotation: Subtype: Link Rect: 196, 157, 233, 167 Annotation: Subtype: Link Rect: 239, 157, 275, 167 Annotation: Subtype: Link Rect: 404, 130, 441, 141 Annotation: Subtype: Link Rect: 447, 130, 483, 141 Annotation: Subtype: Link Rect: 113, 106, 150, 116 Annotation: Subtype: Link Rect: 156, 106, 192, 116 Annotation: Subtype: Link Rect: 231, 67, 268, 78 Annotation: Subtype: Link Rect: 274, 67, 310, 78 Page: Sequence: 24 Annotations: Annotation: Subtype: Link Rect: 479, 743, 516, 754 Annotation: Subtype: Link Rect: 521, 743, 557, 754 Annotation: Subtype: Link Rect: 411, 718, 449, 729 Annotation: Subtype: Link Rect: 455, 718, 491, 729 Annotation: Subtype: Link Rect: 149, 668, 186, 678 Annotation: Subtype: Link Rect: 192, 668, 228, 678 Annotation: Subtype: Link Rect: 183, 629, 220, 639 Annotation: Subtype: Link Rect: 227, 629, 263, 639 Annotation: Subtype: Link Rect: 399, 603, 436, 614 Annotation: Subtype: Link Rect: 442, 603, 478, 614 Annotation: Subtype: Link Rect: 205, 578, 242, 588 Annotation: Subtype: Link Rect: 248, 578, 284, 588 Annotation: Subtype: Link Rect: 122, 553, 160, 563 Annotation: Subtype: Link Rect: 166, 553, 202, 563 Annotation: Subtype: Link Rect: 186, 527, 224, 537 Annotation: Subtype: Link Rect: 230, 527, 266, 537 Annotation: Subtype: Link Rect: 323, 475, 360, 486 Annotation: Subtype: Link Rect: 367, 475, 403, 486 Annotation: Subtype: Link Rect: 426, 450, 464, 461 Annotation: Subtype: Link Rect: 470, 450, 506, 461 Annotation: Subtype: Link Rect: 394, 437, 432, 448 Annotation: Subtype: Link Rect: 438, 437, 474, 448 Annotation: Subtype: Link Rect: 239, 411, 276, 422 Annotation: Subtype: Link Rect: 283, 411, 319, 422 Annotation: Subtype: Link Rect: 387, 386, 425, 397 Annotation: Subtype: Link Rect: 431, 386, 467, 397 Annotation: Subtype: Link Rect: 385, 360, 422, 371 Annotation: Subtype: Link Rect: 428, 360, 464, 371 Annotation: Subtype: Link Rect: 519, 335, 557, 346 Annotation: Subtype: Link Rect: 59, 323, 95, 333 Checksum: 10097d48 Type: CRC32 Checksum: b890d4e6a9a98317cb8e9c909207c04d Type: MD5 Checksum: df1747eb53ca41954cf0adfd9b9903bb7196797e Type: SHA-1 Checksum: 2f614f2c733fecdaa35bb81db89c0d359eb7a66549f5094b6ea6ecfe2bb3f06a Type: SHA-256