Journal of Neurochemistry, 2006, 99, 499–513
doi:10.1111/j.1471-4159.2006.04112.x
Noncompensation in peptide/receptor gene expression and distinct behavioral phenotypes in VIP- and PACAP-deficient mice Beatrice A. Girard,1,* Vincent Lelievre, ,1,2 Karen M. Braas,* Tannaz Razinia, Margaret A. Vizzard,* Yevgeniya Ioffe, Rajaa El Meskini,à Gabriele V. Ronnett,à James A. Waschek and Victor May* *Departments of Anatomy and Neurobiology, Pharmacology and Neurology, University of Vermont College of Medicine, Burlington, Vermont, USA Department of Psychiatry and Mental Retardation Research Center, University of California at Los Angeles, Los Angeles, California, USA àDepartment of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
Abstract Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are closely related neurotrophic peptides of the secretin/glucagon family. The two peptides are derived from a common ancestral gene and share many functional attributes in neuronal development/ regeneration which occur not only from overlapping receptor subtype signaling but also through common mechanisms regulating their expression. Although PACAP or VIP null mice have been generated for study, it is unclear whether the expression of the complementary peptide or their receptor systems are altered in a compensatory manner during nervous system development. By radioimmunoassay and quantitative PCR measurements, we first show that PACAP and VIP have very different temporal patterns of expression in developing postnatal mouse brain. In wild-type animals, PACAP transcript and peptide levels increased rapidly 2- and 5fold, respectively, within 1 week of age. These levels at 1 week of age were maintained through adulthood. VIP tran-
script and peptide levels, by contrast, increased 25- and 50fold, respectively, over a later time course. In parallel studies of development, there were no apparent compensatory increases in brain VIP expression in the PACAP knockout animals, PACAP expression in the VIP-deficient animals, or receptor mRNA levels in either genotype. To the contrary, there was evidence for developmental delays in the expression of peptide and receptor transcripts in the knockout animals. A series of behavioral and neurological tests demonstrated differences between the knockout genotypes, revealing some functional distinctions between the two genes. These results suggest that the PACAP and VIP have evolved to possess distinct biological activities and intimate that the respective knockout phenotypes represent deficits unmitigated by the actions of the complementary related peptide. Keywords: compensatory; pituitary adenylate cyclase-activating polypeptide; peptides; SHIRPA; Phenotype Assessment behavioral assessment; vasoactive intestinal peptide J. Neurochem. (2006) 99, 499–513.
Received February 4, 2006; revised manuscript received June 6, 2006; accepted June 6, 2006. Address correspondence and reprint requests to Victor May, Departments of Anatomy & Neurobiology, and Pharmacology, University of Vermont College of Medicine, 149 Beaumont Avenue, Health Science Research Facility, Room 428, Burlington, Vermont 05405, USA E-mail:
[email protected] James A. Waschek, Department of Psychiatry, University of California at Los Angeles, Mrrc/nrb 345, 635 Charles E. Young Dr South, Los Angeles, CA 90095, USA. E-mail:
[email protected] 1 These authors contributed equally to the work. 2 Current address: INSERM U676, Hoˆpital Robert Debre, 48bd serurier, F-75019, Paris, France.
Abbreviations : cDNA, complementary DNA; CGRP, calcitonin generelated peptide; PAC1, PACAP-selective G-protein-coupled receptor; PACAP, pituitary adenylate cyclase-activating polypeptide; Rn, normalized reporter of quantitative PCR equal to the ratio of the fluorescence emission of the reporter dye, SYBR Green I, and the internal reference dye; DRn, signal magnitude determined as the difference between the Rn value of the sample containing all of the reaction components and that determined under no template control conditions; SHIRPA, (SmithKline Beecham Pharmaceuticals; Harwell, MRC Mouse Genome Centre and Mammalian Genetics Unit; Imperial, College School of Medicine at St. Mary’s; Royal, London Hospital, St. Bartholomew’s and the Royal London School of Medicine; Phenotype, Assessment); VIP, vasoactive intestinal peptide; VPAC, G-protein-coupled receptors exhibiting similar binding affinities for VIP and PACAP.
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Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are related peptides of a superfamily composed of six genes encoding nine bioactive peptides. The members developed from gene/exon duplication and divergence events spanning several hundred million years, resulting in the production of PACAP, VIP, growth hormone releasing hormone, peptide histidine-methionine, secretin, glucagon, glucacon-like peptide-1 and -2, and glucose-dependent insulinotrophic peptide. PACAP is likely the archetypical ancestral peptide and has been highly conserved from protochordates to mammals with one amino acid substitution in the peptide sequence, implying its continued importance through evolution (Sherwood et al. 2000). As PACAP and VIP developed within one branch of the evolutionary scheme, the two peptides are highly related not only in structural but also in biological activities compared to other family members (Vaudry et al. 2000). The PACAP and VIP genes are similar in exon organization (Sherwood et al. 2000). The PACAP precursor molecule is alternatively processed to a-amidated PACAP27 or PACAP38, and PACAP27 is 70% identical to human VIP. By contrast, PACAP27 amino acid homology with glucagon or secretin is more distant (approximately 30% and 33%, respectively). In addition to these homologies, the PACAP and VIP peptides affect function through shared G-proteincoupled receptor subtypes. Only PACAP peptides exhibit high affinity for the PAC1 receptor, whereas VIP and PACAP have similar high affinities for the VPAC1 and VPAC2 receptors (Arimura 1998; Sherwood et al. 2000; Vaudry et al. 2000; Laburthe and Couvineau 2002; Laburthe et al. 2002; Zhou et al. 2002). Compared to VPAC receptors, which appear to be coupled primarily to adenylyl cyclase, PAC1 receptor isoforms display relatively diverse patterns of intracellular signaling, including adenylyl cyclase and phospholipase C, upon PACAP binding. Accordingly, PACAP and VIP may have many overlapping actions as transmitters/modulators and trophic factors in neuronal development, function and regeneration. As examples, PACAP and VIP are hypothalamic peptides capable of stimulating pituitary hormone release, are associated with the retinohypothalamic tract/suprachiasmatic nucleus in circadian rhythm physiology (albeit with different functions), and are found in peripheral autonomic and sensory fibers to affect cardiovascular, gastrointestinal, respiratory, reproductive and urinary functions (Arimura 1998; Sherwood et al. 2000; Vaudry et al. 2000). Moreover, PACAP, VIP and many of their receptor subtypes are expressed early in embryonic central/peripheral nervous system development, and appear to promote neuronal survival, proliferation and fiber outgrowths (Waschek 1995; Shuto et al. 1996; Lu and DiCiccoBloom 1997; Basille et al. 2000; DiCicco-Bloom et al. 2000; Vaudry et al. 2000; Nicot et al. 2002; Waschek 2002; Falluel-Morel et al. 2005). In recapitulation of these developmental trophic functions, both PACAP and VIP have well
established abilities to abrogate apoptotic signals in adult neural cultures after growth factor or serum deprivation, cessation of depolarizing conditions, 6-hydroxy-dopamine and glutamate neurotoxicity, or ischemia (Morio et al. 1996; Uchida et al. 1996; Chang and Korolev 1997; Villalba et al. 1997; Said 2000; Filippatos et al. 2001; Reglodi et al. 2002; Vaudry et al. 2002; Tabuchi et al. 2003). PACAP and VIP are largely expressed by separate neuronal populations in the central and peripheral nervous systems; only fibers in the gastrointestinal tracts demonstrate significant levels of PACAP and VIP colocalization (Fahrenkrug and Hannibal 2004). Similarly, the three PACAP/VIP receptor subtypes demonstrate distinct distribution patterns in brain and peripheral organs (Harmar and Lutz 1994; Vaudry et al. 2000). Several groups have generated PACAP or PAC1 receptor null mice (Jamen et al. 2000; Gray et al. 2001; Hashimoto et al. 2001; Otto et al. 2001b; Colwell et al. 2003; Colwell et al. 2004). Although there may be variability in mouse phenotypes among laboratories stemming from differences in knockout methodologies and genetic backgrounds, the PAC1 receptor and PACAP knockout animals share some defects, including hyperactive psychomotor behavior, poor fertility, circadian rhythm irregularities and abnormalities in glucose/lipid homeostasis. Furthermore, PACAP null mice have been reported to exhibit high rates of postnatal mortality (> 60%), which may be related to acute thermosensitivity and apnea (Gray et al. 2002; Cummings et al. 2004). The recently developed VIP knockout mice may present some infertility but, like the PACAP knockouts, demonstrate clear circadian defects (Colwell et al. 2003; Aton et al. 2005). Given the strong functional overlap between PACAP and VIP, there have been suggestions that the viability of the PACAP or VIP knockout animals may be related to the compensatory actions of complementary VIP and PACAP peptides, respectively. There are many recent examples for compensatory mechanisms to ameliorate the phenotype of knockout animals, including genes regulating cell cycle, apoptosis, tissue morphogenesis, and cellular neuropeptide, receptor and channel expression (Zheng et al. 2000; Geng et al. 2001; Tong and Pollard 2001; Tse et al. 2001; Deighan et al. 2004; Takahashi et al. 2004; Wan et al. 2005). To examine whether there are similar compensatory changes in these peptide null animals, we assessed changes in the expression of PACAP, VIP and their receptor subtypes in the brains of the VIP and PACAP knockout animals during different stages of postnatal development. A standardized series of simple neurological and behavioral tests was performed to demonstrate differences between the knockout genotypes. As the PACAP and VIP peptidergic systems appear distinct, the phenotypes in the PACAP or VIP null animals most likely represent physiological deficits unabated by the actions of their related peptides.
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Materials and methods Animals PACAP- and VIP-null animals were generated in a pathogen-free facility at UCLA and backcrossed for at least six generations with C57BL/6 mice as described previously (Colwell et al. 2003, 2004). The PACAP- and VIP-deficient animals used in all studies were physically sound but demonstrated an overall reduction in body weight of about 10% and 15%, respectively, when compared to control animals (data not shown); the small weight reduction was accompanied by parallel changes in body length. The physiological basis for the observed changes in overall growth in the knockout animals remains to be elucidated, but may be related to peptide effects on food intake, nutritional absorption metabolism and/or growth. Radioimmunoassay Mice (mixed sex) from different postnatal developmental stages were killed by decapitation and the brains (including cerebellum) removed for mid-sagittal hemisection. After genotyping, one-half of the brain was extracted in 5 N acetic acid with 0.3 mg/mL phenylmethylsulfonyl fluoride using a ground glass-ground glass homogenizer. An aliquot was removed for total protein assay using the bicinchoninic acid (BCA) reagent. The homogenates were frozen and thawed three times, and centrifuged at 1000 · g to remove debris; the resulting extracts were lyophilized and subsequently reconstituted in 100 mM sodium phosphate buffer, pH 7.5, containing 1% Triton X-100 and phenylmethylsulfonyl fluoride for double antibody radioimmunoassays, as described previously using 1 : 30 000 anti-PACAP38 (Peninsula Laboratories, Belmont, CA, USA) or 1 : 90 000 anti-VIP (Ab 7913, Dr. J. Walsh, UCLA) (Brandenburg et al. 1997; Braas and May 1999; Girard et al. 2002). Each sample was assayed over a 10-fold dilution range to ensure data interpolation within the linear segment of the standard curves. Assay midpoint for PACAP38 and VIP was 3.5-5 fmol. In direct assessments, each antiserum failed to demonstrate detectable crossreactivity with related peptide members under these assay conditions. PACAP38 peptides in PACAP knockout mice brains were undetectable under current assay conditions. As brain PACAP38 levels are typically 100-fold greater than those for PACAP27, only PACAP38 levels were determined. A residual < 2% VIP-immunoreactive material of unknown origins was apparent in brains from VIP knockout animals. Brain PACAP and VIP levels in respective adult heterozygote animals were 46-50% of wild-type levels. Three to five individual samples were measured for each group at each developmental period. Quantitative PCR measures of transcript levels The complementary half of the hemisected brain was homogenized in Stat-60 total RNA/mRNA isolation reagent (Tel.Test ‘B’, Friendswood, TX, USA) as described previously (Braas and May 1999; Girard et al. 2002; Girard et al. 2004). The RNA (2 lg) was used to synthesize first strand complementary DNA (cDNA) using SuperScript II reverse transcriptase and random hexamer primers with the SuperScript II Preamplification System (Invitrogen, Carlsbad, CA, USA) in a 20-lL final reaction volume. All RNA in the developmental series was reverse transcribed simultaneously to obviate variability. Following the reverse transcriptase reaction,
the cDNA samples were treated with RNase H to remove residual RNA. The preparation of quantitative PCR standards for PACAP and VIP transcripts was described previously (Girard et al. 2002). To prepare real-time quantitative PCR standards for the PACAP/VIP receptors, the amplified PAC1, VPAC1 and VPAC2 cDNA products were ligated directly into pCR2.1 TOPO vector using the TOPO TA cloning kit (Invitrogen). The nucleotide sequences of the inserts were verified by automated fluorescent dideoxy dye terminator sequencing (Vermont Cancer Center DNA Analysis Facility). To estimate the relative expression of the receptor transcripts, 10-fold serial dilutions of stock plasmids were prepared as quantitative standards. The range of standard concentrations was determined empirically. Real-time quantitative PCR was performed essentially as described using SYBR Green I detection (Girard et al. 2002, 2004). Complementary DNA templates, diluted 5-fold to minimize the inhibitory effects of the reverse transcription reaction components, were assayed using SYBR Green I JumpStartTM. Taq ReadyMix (Sigma, St. Louis, MO, USA) containing 3.5 mM MgCl2, 200 lM dATP, dGTP, dCTP and dTTP, 0.64 U Taq DNA polymerase and 300 nM of each primer in a final 25-lL reaction volume. The realtime quantitative PCR was performed on an ABI Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA) using the following standard conditions: (i) serial heating at 50C for 2 min and 94C for 2 min; (ii) amplification over 40 cycles at 94C for 15 s and 62C for 40 s. The amplified product from these amplification parameters was subjected to SYBR Green I melting analysis by ramping the temperature of the reaction samples from 62C to 95C. A single DNA melting profile was observed under these dissociation assay conditions demonstrating amplification of a single unique product free of primer dimers or other anomalous products. Oligonucleotide primer sequences are: PAC1 receptor (S) 5¢-AACGACCTGATGGGACTAAAC-3¢ (AS) 5¢-CGGAAGCGGCACAAGATGACC-3¢; VPAC1 receptor (S) 5¢-CGGCCACCCGACATTGGGAAG-3¢ (AS) 5¢-CTGCATGTGGCGCCGTTGCTG-3¢; VPAC2 receptor (S) 5¢AATGACCAGTCACAGTACAAGA-3¢ (AS) 5¢-TCACACTGTACCTCACTGTTCA-3¢; PACAP (S) 5¢-CATGTGTAGCGGAGCAAGGTT-3¢ (AS) 5¢-GTCTTGCAGCGGGTTTCC-3¢; VIP (S) 5¢TTGGCAAACGAATCAGCAGTAG-3¢ (AS) 5¢-ATTTGCTTTCTAAGGCGGGTGTA-3¢. The oligonucleotide primers for PACAP and VIP were validated previously (Girard et al. 2002). The melting profiles for amplified PAC1, VPAC1 and VPAC2 receptor DNA fragments are shown in Fig. 1, verifying the unique product amplification in the quantitative PCR assays. For data analyses, a standard curve was constructed by amplification of serially diluted plasmids containing the receptor target sequence. Data were analyzed at the termination of each assay using the Sequence Detector 1.7a software (Applied Biosystems). In standard assays, default baseline settings were selected. The increase in SYBR Green I fluorescence intensity (DRn) was plotted as a function of cycle number and the threshold cycle was determined by the software as the amplification cycle at which the DRn first intersects the established baseline. The PAC1 and VPAC receptor transcript levels in each sample were calculated from the threshold cycle by interpolation from the
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(a)
(b)
(c)
Fig. 1 Unique quantitative PCR products amplified from PAC1, VPAC1 or VPAC2 receptor primers. The uniqueness of the amplicons synthesized using the PAC1 (a), VPAC1 (b) and VPAC2 (c) primers and amplification conditions were assessed by SYBR Green I dye melting analyses. Ramping of the temperature from 65C to 95C produced a single unique DNA dissociation curve in each case. The amplification conditions did not generate multiple or anomalous products, or primer dimers. Blue tracing line, sample extract template; green tracing line, no template control.
standard curve to yield the relative changes in expression. Data were typically normalized to 18S RNA levels. For each target sequence, all samples were amplified together in the same assay to minimize variability. All assays were repeated two or three times to verify data reproducibility. As for peptide transcript measurements, there were three to five samples for each group at each developmental stage.
SHIRPA tests At 6 weeks of age, animals were weaned, tagged and genotyped. Two-month-old PACAP- or VIP-knockout animals were then transferred to the UCLA Behavioral Testing Core and housed for 2 weeks prior to start of SHIRPA (SmithKline Beecham Pharmaceuticals; Harwell, MRC Mouse Genome Centre and Mammalian Genetics Unit; Imperial College School of Medicine at St. Mary’s; Royal London Hospital, St. Bartholomew’s and the Royal London School of Medicine; Phenotype Assessment) tests (Rogers et al. 1997, 2001; for more information, see http://www.mgu.har.mrc.ac.uk/facilities/mutagenesis/mutabase/shirpa_summary.html). Housing conditions (light/dark cycle, temperature, access to food and water) were similar at all facilities; animal body weight and sex were carefully monitored before each series of tests. Every animal was tested four times to minimize any behavioral artefacts that may have occurred randomly. For each strain, the same responses were observed for each behavior assessment or task, regardless of sex. To reduce errors within groups due to subtle increases in response magnitude in female mice, data from males and females were analyzed separately. The same behavior assessments in males and females were statistically different but, to avoid redundancy, only data from male mice are presented. For initial screening, a battery of 30 tests was performed. Animals were first transferred into a viewing jar for a 4-min observation (spontaneous activity, defecation and urination), and then relocated in the working area for motor behavior (walking distance, grip strength, balance and coordination, righting reflex) and sensory activity (visual placing, startle response, touch escape, toe pinch, transfer arousal) measurements. Finally, animals were tested for some neuropsychiatric or psychological deficiencies (irritability, fear, aggression and vocalization). To further assess the differences in locomotor functions between wild-type and knockout animals, mice were transferred into open Plexiglas computerized-recording arenas that measured 10.75 inches square and 8 inches high for real-time movement assessments using infrared sensors (Model ENV-510, MED Associates, St. Albans, VT, USA). Animal activity tracking in the XY-plane was monitored through 16 evenly spaced infrared sensors; additional photobeam detectors were placed 2 inches above the floor of the apparatus to detect hind-leg vertical rearings in the z-axis. Cage mates were transported to the dimly illuminated testing area and loaded immediately into individual arenas for 10-min activity trials in the absence of personnel in the room. The four activity chambers in the room allowed multiple animals to be tested simultaneously. The instrumentation and MED Associated Activity software allowed detailed measures of locomotor activities including total distance traveled, resting time and movement speed. Statistical analyses All values are expressed as the mean ± SEM. Data among groups were analyzed by 2-factor ANOVA, with genotype and age as the two factors; all tests were followed by Student-Newman-Keuls or HolmSidak posthoc analyses and p-values less than 0.05 were considered significant. Calculations were performed using SAS Systems 9.1.2 (SAS Institute, Cary, NC, USA), SIGMASTAT 3.0 and SIGMAPLOT 8.0 (SPSS, Inc., Chicago, IL, USA) software. For SHIRPA analyses, animals were assessed using a uniform scoring system to measure overall performance. Specific details on ranking are given in Table 1. For the four independent assessment
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Table 1 SHIRPA tests Mouse strain Evaluation Viewing Jar Body position Activity Respiration rate Tremor Excrement Open Field Transfer arousal Activity Eye closure Coat status Startle response Gait Pelvic elevation Tail elevation Touch escape Position passivity Tail Lifting Limb grasp Visual placing Grip strength Body tone Horizontal Grid Pinna reflex Corneal reflex Toe pinch Horizontal Wire Wire maneuver Supine Restrain Skin color Heart rate Abdomen tone Lacrimation Salivation Provoked biting Righting reflex Tube Contact righting Vertical Grid Negative geotaxis Handling Fear Irritability Aggression Vocalization
Scoring index
0 0 0 0 0
C57BL/6 (wild-type; n ¼ 9)
PACAP )/) (n ¼ 8)
(flat) – 5 (leaps) (resting) – 4 (restlessness) (gasping) – 3 (hyperventilation) (none) – 2 (marked) (none) – 2 (urine/feces)
2.94 1.08 2.0 0.12 1.30
± ± ± ± ±
0.03 0.05 0 0.09 0.21
3.08 1.21 2.0 0.13 0.80
± ± ± ± ±
0.05* 0.10 0 0.05 0.28
0 (coma) – 5 (frantic excitation) Squares crossed in 30 s 0 (open) – 2 (closed) 0 (normal) – 1 (piloerection) 0 (none) – 3 (high jumps) 0 (normal) – 3 (incapacity) 0 (very flattened) – 3 (elevated) 0 (dragging) – 2 (straub tail) 0 (none) – 3 (anticipated/vigorous) 0 (tail hold) – 4 (no struggle)
1.88 14.58 0 0 0 0 2.06 1.01 1.66 0.15
± ± ± ± ± ± ± ± ± ±
0.34 1.26 0 0 0 0 0.04 0.02 0.08 0.06
3.25 27.28 0 0 0 0 2.33 1.34 2.33 0.8
± ± ± ± ± ± ± ± ± ±
1.00 3.0 2.92 0.92
± ± ± ±
0 0 0.05 0.05
1.00 3.0 3.32 0.88
± ± ± ±
0 0 0 0
(none) – 1 (present) (none) – 4 (extended anticipation) (none) – 4 (unusually strong) (flaccid) – 2 (resistant)
VIP )/) (n ¼ 9)
3.07 1.32 2.0 0.13 0.93
± ± ± ± ±
0.04* 0.13 0 0.09 0.14
0.44* 1.91*, 0 0 0 0 0.04 0.11* 0.61 0.17
3.15 22.10 0 0 0 0 2.19 1.00 2.05 0.15
± ± ± ± ± ± ± ± ± ±
0.24* 0.37* 0 0 0 0 0.09 0.00 0.23 0.11
0 0 0.09*, 0.06
1.00 3.0 2.43 0.75
± ± ± ±
0 0 0.14* 0.05
0 (none) – 2 (repeated flick) 0 (none) – 2 (multiple eye blinks) 0 (none) – 4 (multiple leg extension)
1.00 ± 0 1.00 ± 0 2.19 ± 0.17
1.00 ± 0 0±0 2.46 ± 0.22
1.00 ± 01.0 1.00 ± 0 2.32 ± 0.24
0 (active grip with hindleg) – 4 (fall)
0.21 ± 0.08
0.21 ± 0.12
2.16 ± 0.34**
0 0 0 0 0 0 0
1.00 1.00 0.80 0 0 0 0
1.00 1.00 0.80 0 0 0.34 0
1.00 1.00 0.82 0 0 0 0
(blanched) – 2 (red) (slow) – 2 (fast) (flaccid) – 2 (resistant) (none) – 1 (present) (none) – 2 (wet) (none) – 1 (present) (normal) – 2 (land on back)
± ± ± ± ± ± ±
0 0 0.08 0 0 0 0
± ± ± ± ± ± ±
0 0 0.04 0 0 0.09** 0
± ± ± ± ± ± ±
0 0 0.09 0 0 0 0
0 (absent) – 1 (present)
1.00 ± 0
1.00 ± 0
1.00 ± 0
0 (turn/climb) – 4 (fall off)
0.03 ± 0.03
1.78 ± 0.20**
0.06 ± 0.04
0 0 0 0
0.92 0.92 0 0.71
0.46 0.91 0 0.27
0.89 1.00 0 0.70
(none) (none) (none) (none)
– – – –
1 1 1 1
(arousal freezing) (supine struggle) (provoked biting) (when handled)
± ± ± ±
0.05 0.05 0 0.10
± ± ± ±
0.10** 0.05 0 0.10**
± ± ± ±
0.07 0 0 0.09
* Statistically different from wild-type C57BL/6 at p < 0.05. Statistically different from VIP )/) at p < 0.05. ** Statistically different from both strains at p < 0.05.
series, data (mean ± SEM) were compared between animals and among groups. Because the SHIRPA tests were repeated four times over a 1-month period, the contribution of time or learning to the
task performance was determined; the possible interactions between time and groups for a given task were also investigated. Student’s t-test, ANOVA or covariance analyses were performed followed by ad-
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hoc postrun tests to assess statistical significance among groups or association of variable, such as genotype and time.
Results
PACAP and VIP display differential patterns of expression in developing postnatal brain For direct comparative assessments, the same brain sample extracts were measured for PACAP and VIP peptide levels by radioimmunoassay. The adult C57BL/6 mouse brains contained approximately 120 fmol PACAP38/mg protein and 500 fmol VIP/mg protein, which agreed well with previous data in rodents to validate our current measurements (Huang et al. 1986; Arimura et al. 1991; Tatsuno et al. 1994). Brain PACAP38 level in postnatal day 1 (P1) wild-type animals was 24.1 ± 4.6 fmol/mg protein and was approximately 2.5fold higher than that for VIP (9.4 ± 1.8 fmol VIP/mg protein) at the same postnatal age. The levels for PACAP subsequently increased sharply more than 5-fold by P7 to concentrations that were sustained through adulthood (Fig. 2a, open bars). These were comparable to patterns from previous work (Tatsuno et al. 1994; Shuto et al. 1996). The corresponding developmental changes in PACAP mRNA levels in the contralateral brain hemispheres were measured by quantitative PCR and shown to be less pronounced, increasing only 2-fold from birth (P0) to P7 (Fig. 3a, open bars). The early postnatal PACAP gene expression between the first and second week, before a modest but significant decline to adult levels by 1 month of age, may be relevant to the putative actions of PACAP on cerebellum granule development (Gonzalez et al. 1996) and/ or myelinogenesis (Lee et al. 2001). In contrast to the early and modest changes in PACAP expression, the increase in brain VIP peptide levels during postnatal development of C57BL/6 mice was delayed and greater in magnitude, ultimately attaining a 50-fold induction by adulthood (Fig. 2b, open bars). After an apparent initial lag period (P1-P4) brain concentrations of VIP increased more than 10-fold from 9.4 ± 1.8 to 108 ± 16 fmol VIP/mg protein by P7. After P10, the levels of brain VIP increased steadily to more than 500 fmol VIP/mg protein by P28, which represented adult levels. These developmental changes in brain VIP peptide were mirrored completely by a 25-fold increase in VIP transcript levels from P1 to adulthood (Fig. 3b, open bars). PACAP/VIP receptor subtypes demonstrate different developmental expression patterns The same cDNA templates for quantitative PCR measures of peptide transcript levels were also used to assess the correlative changes in transcript expression of the three PACAP/VIP receptor subtypes during development. As described in Materials and methods, the amplification
Fig. 2 Radioimmunoassay of brain PACAP38 and VIP content in C57BL/6, PACAP -/- and VIP -/- mice during postnatal development. Brain tissues from mice of different developmental ages were homogenized in acetic acid with protease inhibitors as described in Materials and methods. An aliquot of the extract was removed for protein measurements; the remaining samples were lyophilized and resuspended in assay buffer for PACAP38 (a) and VIP (b) radioimmunoassay. Data represent mean fmol peptide/mg protein from 3-5 animals ± SEM. h, C57BL/6; , VIP )/); j, PACAP )/). Expression levels in P1 wild-type shown in open hatched bar for ease of data comparisons. BLD, below level of detection. From 2-way ANOVA (independent factors were genotype and age), p-values for genotype, age and genotypeage interaction were < 0.001 for all variables measured. *, statistically different from wild-type postnatal day 1 (P1) at p < 0.05. +, statistically different from wild-type of the same postnatal age at p < 0.05.
primers and parameters for each of the three receptor subtypes generated a single product with unique melting isotherm to validate the quantitative procedures (Fig. 1). Unlike many other receptor systems, the expression of PAC1 receptor transcripts did not appear to be regulated during the postnatal periods. The PAC1 receptor transcript levels were already high in brains of C57BL/6 mice at birth and the levels, normalized to tissue 18S levels, did not change
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those for VIP appear more closely aligned with the expression of VPAC1 receptors in the CNS.
Fig. 3 Postnatal brain PACAP and VIP transcript expression in different mouse strains. Contralateral halves of mouse brains described in Fig. 2 were processed for total RNA; 2 lg total RNA was reversed transcribed using random primers for quantitative PCR measurement of PACAP (a) and VIP (b) mRNA levels as described in Methods. PACAP transcript levels in PACAP )/) animals were within assay background. Disrupted mutant VIP transcripts detected in VIP )/) animals did not encode peptide and were not measured. Data represent mean percent change compared to P1 wild-type levels from 3 to 5 animals ± SEM. h, C57BL/6; , VIP )/); j, PACAP )/). Expression levels in P1 wild-type shown in open hatched bar for ease of data comparisons. ND, not determined. For the dependent PACAP transcript variable, 2-way ANOVA p-values for genotype, age and genotypeage interaction were < 0.001, < 0.01 and < 0.05, respectively. For the VIP transcript variable, 2-way ANOVA p-values for genotype, age and genotype-age interaction were all < 0.001. *, statistically different from P1 wild-type at p < 0.05. +, statistically different from wild-type of the same postnatal age at p < 0.05.
appreciably throughout postnatal and adult development (Fig. 4a, open bars). Similar to previous work, these results suggested that PAC1 receptor signaling is already a significant regulator of neuro/oligogenesis and/or differentiation during fetal development (Masuo et al. 1994). Unlike PAC1 receptor mRNA levels, both VPAC1 and VPAC2 receptor brain transcript levels were increased postnatally (Fig. 4b,c, open bars). While VPAC2 receptor mRNA levels increased maximally 2-fold from birth through adulthood, the increase in VPAC1 transcript expression was more dramatic, exceeding 10-fold over the same postnatal periods. Accordingly, in comparisons with peptide levels above, the developmental changes in brain PACAP expression may be better correlated with PAC1 and VPAC2 receptor expression patterns whereas
Apparent lack of compensatory peptide expression in PACAP- and VIP-deficient mice Related genes of the same family with similar or overlapping functions in some cases are thought to supplant and ameliorate physiological defects in knockout studies. As PACAP and VIP emerged from gene duplication and divergence events, and can potentially be regulated by similar sets of factors for shared functions, there was some anticipation that similar compensatory mechanisms by the complementary peptide may be present to blunt or mask the physiological phenotypes in the knockout animals (Sherwood et al. 2000). As a first approximation to evaluate whether these compensatory processes were engaged, changes in VIP expression were assessed in brains of PACAPknockout animals; conversely, PACAP levels were measured in the VIP-deficient mice. If peptide compensatory mechanisms were activated in these knockout animals during development, the responses were anticipated to be manifested as increased peptide expression beyond normal levels observed in wild-type animals. As changes in peptide production are frequently mirrored by changes in transcript levels, both parameters were measured in the same tissue samples over postnatal development as a means of confirming independently the validity of the observations. There were no apparent long-term compensatory mechanisms in either of the peptide-deficient animals. Homologous PACAP peptides and transcripts were undetectable in the PACAP knockout animals. When VIP peptide content was measured in brains of the PACAP knockout animals, the VIP levels were not appreciably changed compared to wild-type animals of the same early postnatal age (P1 - P4; Fig. 2b, black bars). By contrast, at P7 when VIP production levels are accelerated, there was no acceleration in PACAP knockout mice. Moreover, VIP content in PACAP knockout mice at P7 was 30% of that in wild-type animals (Fig. 2b). These decreases were paralleled by a 50% diminution in VIP mRNA levels in the same brain tissues (Fig. 3b, black bars). This suggests an interruption or developmental delay in VIP expression by P7 in PACAP knockout mice. The deficits in VIP content were corrected by P10 and at the later postnatal ages examined, VIP content and transcript levels in brain tissues of PACAP knockout animals did not vary more than 20% of control wild-type values. PACAP also failed to compensate for VIP and was developmentally delayed in the VIP-deficient animals. The levels of PACAP38 immunoreactive material in the brains of P1-P4 VIP-deficient and C57BL/6 animals were comparable (Fig. 2a, gray bars), but by P7, when PACAP38 content in wild-type animals increased sharply, PACAP38 peptide levels in the VIP knockouts were not apace and represented only 40% of wild-type brain levels from the same postnatal
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were decreased 35% compared to their wild-type counterparts (Fig. 3a, gray bar). Unlike patterns for VIP expression in PACAP knockouts, however, the deficiencies in PACAP transcript levels in the VIP knockouts were not quickly rectified over time. As PACAP mRNA levels declined over the course of normal postnatal brain development, PACAP transcripts in the VIP knockout mice were consistently decreased 30-40% compared to wild-type levels until P49 (Fig. 3a, gray bar).
Fig. 4 Brain PAC1, VPAC1 and VPAC2 receptor transcript levels in wild-type and mutant mouse strains. Brain cDNA templates from Fig. 3 were also submitted for quantitative PCR PAC1 (a), VPAC1 (b) and VPAC2 (c) receptor mRNA measurements. Data represent mean percent change compared to P1 wild-type levels from 3 to 5 animals ± SEM. h, C57BL/6; , VIP )/); j, PACAP )/). Expression levels in P1 wild-type shown in open hatched bar for ease of data comparisons. ND, not determined. For each dependent variable, 2-way ANOVA p-values for genotype, age and genotype-age interaction were as follows: PAC1, < 0.001, < 0.002 and < 0.001, respectively; VPAC1, < 0.001, < 0.001 and < 0.005, respectively; and VPAC2, < 0.001, < 0.04 and < 0.001, respectively. *, statistically different from P1 wildtype at p < 0.05. +, statistically different from wild-type of the same postnatal age at p < 0.05.
age (Fig. 2a, gray bar). After the delay, brain PACAP38 content in the VIP-deficient animals increased and was comparable to wild-type levels for the remaining postnatal periods. To mirror the changes in peptide content, PACAP transcript levels in the brains of P7 VIP-deficient animals
PACAP and VIP receptor subtype expression is diminished in brains of PACAP- or VIP-deficient mice The regulation of the three PACAP/VIP receptor subtypes in the central and peripheral nervous systems is not well understood. In many physiological situations, however, the expression of peptides and their receptors follows a reciprocal relationship (Moller et al. 1997; Zhang et al. 1998; Zhou et al. 1999). Injury-mediated inductions of PACAP and/or PACAP mRNA expression in facial motor neurons and autonomic ganglia neurophenotypic plasticity, for example, are correlated with diminished PAC1 receptor transcript expression, suggesting that enhanced PACAP signaling may down-regulate the levels of the cognate receptor (Moller et al. 1997; Zhou et al. 1999). Accordingly, it is unclear whether deficiencies in peptide expression in the knockout animals are met by altered receptor expression. The expression of the different PACAP and VIP receptor may be regulated independently of peptide levels and hence be unaffected in the knockout animals compared to wild-type controls of the same developmental age; alternatively, specific PACAP/VIP receptor subtype expression may be augmented as a consequence of peptide deletion. Contrary to expectations, the levels of all three receptor subtype transcripts, were diminished in the PACAP and VIP knockout animals compared with their wild-type counterparts (Fig. 4). However, for each receptor subtype, the patterns for decreased receptor transcript expression appeared different. Unlike the stable expression levels for PAC1 receptor transcripts in wild-type animals during postnatal development, PAC1 receptor transcript levels were diminished approximately 50% by P14-P28 in the PACAP or VIP knockout animals (Fig. 4a, black and gray bars, respectively). The increase in brain VPAC2 receptor transcripts in late developing wild-type animals was not evident in the VIP and PACAP knockout animals; on the contrary, VPAC2 receptor transcripts in P28 PACAP knockout animals was only 20% of norm (Fig. 4c, black bars). Relatedly, the developmental increase in brain VPAC1 receptor transcript expression appeared delayed after P4 in the PACAP or VIP knockout animals such that VPAC1 transcript levels were diminished 15-40% at subsequent ages (Fig. 4b). While these results may mitigate arguments for a generalized mechanism for altered PAC1/VPAC receptor expression in the knockout animals, these results may have stemmed in part from
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neuronal developmental delays or defects from loss of trophic neuropeptide availability. PACAP or VIP knockout animals have altered locomotor behavior In initial assessments, when the animals were placed in a viewing jar for 4 min for observations in spontaneous activity, there were minor nonsignificant differences among strains in some of the basic voluntary (sleeping, resting, sitting or walking) and involuntary (defecation, urination, shivering and tremor) activities. As shown in Table 1, these tests revealed that only the overall body position was significantly different in both knockout animals when compared to control age-matched wild-type animals. Whereas wild-type mice were mostly sitting and resting during the 4-min observation period, the PACAP- or VIPdeficient mice explored the viewing jar and spent more time rearing (Table 1, Viewing Jar, Activity and Body Position parameters). Also, the knockout animals appeared to demonstrate more locomotor activity (see below), the activity assessments in this group of tests encompassed other parameters including scratching and grooming, which tempered the differences between wild-type and knockout animals from significance. Although not statistically different, knockout animals also appeared to urinate and defecate less than wild-type controls. The second set of tests, performed in an open field for locomotor behavior, clearly revealed some of the principal differences among strains (Table 1, Open Field, Activity). Whereas no significant differences were noted in degree of eye closure, gait or startle responses, there were important distinctions in transfer arousal and locomotor activity tasks between wild-type and knockout animals. Both knockout animals strains exhibited increased locomotor activities, suggesting that the lack of either VIP or PACAP expression leads to less anxiety and/or hyperkinetic disorder. Whereas the control wild-type animals displayed brief but marked absence of movement after transfer into a new environment, the VIP- or PACAP-deficient males very quickly explored the open field (Table 1). This behavior was amplified with striking increases in locomotor activity and exploration in the open field arena (Fig. 5). In analyzing this complex behavior, the animals were transferred to a computer-controlled arena to measure the individual parameters of locomotor activity. These parameters, which included total walking distance and resting time, were measured during the 10-min running period and the study was repeated four times within a month. As shown in Fig. 5a, during the first few minutes of the trial, the PACAP or VIP knockout animals demonstrated similar alterations in locomotor behavior compared to wild-type controls; both knockout animals traveled greater total distance, moved faster and rested less than wild-type mice. At these trial periods, the ambulatory distance traveled for PACAP-/- or VIP-/- animals were different from wild-type
mice (Table 2). However, at later times during the same 10 min trial period (Fig. 5b), activity differences between PACAP- and VIP-deficient mice became apparent. Whereas VIP knockout mice, as in control wild-type animals, showed a reduction in ambulatory distance traveled over time, the PACAP knockout mice maintained an elevated rate of locomotor activity (Fig. 5b). The distance traveled for PACAP -/- animals during later trial periods remained elevated and different from wild-type mice; activity of VIP -/- mice was not different from wild-type mice (Table 2). The difference reflected in part an increase in resting time in the VIP knockout animals, whereas the PACAP knockout mice demonstrated no variations in resting intervals from the first to the last minute of recording (Table 2). PACAP and VIP knockout animals demonstrate differences in psychomotor and sensorimotor tasks In addition to locomotor activity, the knockout animals were also assessed in psychomotor tasks including sensory responses (touch escape, position passivity and visual placing), aggressive behavior (biting reflex and escape), and balance and coordination (wire maneuver, righting reflex and somersault). These tasks also revealed important behavioral differences between PACAP- and VIP-deficient animals. Firstly, as shown in Table 1, PACAP knockout mice differed from both wild-type and VIP null animals by being more aggressive and/or less fearful, as revealed in several different measures, including transfer arousal, fear index and vocalization upon handling. Furthermore, PACAP knockout mice displayed atypical negative geotaxis behavior (Table 1). When the animals were placed on a horizontal grid, which was subsequently raised to a vertical with the animal facing the floor, the PACAP gene-disrupted mice not only lacked the conventional reflex to turn and climb the grid (typically indicative of vestibular and/or proprioceptive sensory defects), but also frequently escaped from the apparatus onto the laboratory bench. Secondly, unlike the PACAP null animals, the VIP knockout mice showed significant impairment in muscular strength. This was particularly evident in the grip strength and wire maneuver tasks; in these cases, the VIP knockout mice showed significant reduction in grip strength as well being unable to grasp the wire with their hindlegs (Table 1). These defects may have contributed to the reductions in ambulatory activity and body tone observed previously. Discussion
From many perspectives, there are important similarities between the PACAP and VIP peptidergic systems. Both genes stemmed from a common branch of a molecular cladistic tree that gave rise to other related peptides including secretin, glucagon, glucagon-like peptides and glucose-
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Fig. 5 Arena locomotor activity of wild-type and knockout mice. Adult (2-month-old) mice exploratory activities in an open arena were recorded for 10 min; the representative activities of the 2-4 min (a) and the 8-10 min (b) time periods among the three strains are shown. During the initial periods of the recording session, the knockout ani-
mals were more active and spent more time in the center of the arena than wild-type controls, suggesting less anxiety. In the latter part of the same sessions, the distance traveled by VIP )/) and C57BL/6 animals diminished whereas the PACAP )/) mice maintained a higher level of activity. Representative data of six animals per group.
Table 2 Arena activity
Distance traveled (cm)
Resting time(s)
Mouse strain
2–4 min
8–10 min
2–4 min
8–10 min
C57BL/6 (n ¼ 6) PACAP )/) (n ¼ 6) VIP )/) (n ¼ 6)
148.4 ± 11.5 204.4 ± 7.8* 211.7 ± 13.1*
121.4 ± 8.9 209.2 ± 14.0* 148.8 ± 13.1
37.2 ± 1.3 33.1 ± 2.3 28.7 ± 4.3*
43.5 ± 1.4 34.5 ± 1.0* 40.3 ± 1.6 +
2-way ANOVA p-values for genotype, time and genotype · time were < 0.001, < 0.001 and < 0.002, respectively. * Statistically different from wild-type C57BL/6 for the same period (within column) at p < 0.05. Statistically different from 2 to 4 min VIP )/) activity period at p < 0.05.
dependent insulinotrophic peptide. Accordingly, PACAP and VIP have shared derived characteristics including peptide sequence, binding to receptor subtypes, and regulated expression and function in a variety of physiological systems (Sherwood et al. 2000). Both peptides produce dose-dependent vasodilation in a variety of vessels, and are well studied anti-inflammatory mediators (Naruse et al. 1993; Ganea and Delgado 2002; Dalsgaard et al. 2003). But PACAP and VIP have gained special prominence in neurodevelopment. PACAP and PAC1 receptor transcripts are expressed during early embryogenesis (E10 or earlier in mouse), at complementary sites within proliferative zones of the developing
nervous system (Tatsuno et al. 1994; Shuto et al. 1996; Waschek et al. 1998). PACAP and/or VIP have been shown to promote the survival of autonomic, cortical, hindbrain, cerebellar and sensory neurons, and to facilitate either proliferation or differentiation depending on neuronal identity and growth factor context (Waschek 2002). They are neuroprotective to cortical, hippocampal, septal, mesencephalic, sympathetic and dorsal root ganglion neurons upon toxicity challenges or growth factor withdrawal by abrogating apoptotic signaling pathways (Vaudry et al. 2000; Zhou et al. 2002). PACAP and VIP can be regulated in parallel in vivo and in vitro. In primary neuronal culture, for example,
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depolarization, cytokines and cyclic AMP mechanism can augment peripheral neuron PACAP and VIP expression (Brandenburg et al. 1997; Girard et al. 2002); in classical axotomy paradigms, both peptides are up-regulated in injuryinduced neuroplasticity (Moller et al. 1997; Zhou et al. 1999; Jongsma Wallin et al. 2001). Accordingly, the parallels between PACAP and VIP expression and function are considerable. Given these attributes, the PACAP and VIP have been considered frequently as alternative peptides in a redundant or fail-safe mechanism to assure appropriate physiological development and function. But if the developmental regulators of PACAP and VIP expression were shared, then the compensatory responses in PACAP or VIP expression could present challenges in interpreting the phenotype of mice with targeted disruption of the complementary peptide gene. Several developmental, metabolic and behavioral phenotypes have been described for PACAP knockout animals including high early postnatal mortality (> 60%) which may be related to abnormal lipid and glucose handling, impaired adaptive thermogenesis, stress intolerance, reduced respiratory responses and apnea (Sherwood et al. 2000; Gray et al. 2001; Gray et al. 2002; Cummings et al. 2004). As PACAP and PAC1 receptor expression in neural tissues is prominent during early embryogenesis, the ability for PACAP null mice to survive, especially under more optimal conditions, has been attributed in part to compensatory VIP expression (Sherwood et al. 2000). Much less is known about neurological and behavior deficits in VIP knockout animals and whether their viability is related to compensatory PACAP expression. There are many examples of compensatory mechanisms mitigating phenotype expression in knockout studies of diverse physiological processes, such as cell cycle progression, tissue development, and receptor/channel function. In cyclinD1 knockouts, for example, cyclinD3 is upregulated to levels approaching those for cyclinD1(Geng et al. 2001; Tong and Pollard 2001), and caspase-9 or caspase-3 deficiency results in increased cellular caspase activation (Zheng et al. 2000). The compensatory roles of Foxa1 and Foxa2 mask defects in lung morphogenesis in single gene knockouts (Wan et al. 2005), a1-adrenoceptors can substitute for one another in specific adrenoceptor subtype deletions (Deighan et al. 2004), related ORL1 receptors compensate for losses in opioid receptor expression (Clarke et al. 2002) and CaV2 channel members can compensate for deficiencies in a1B subunit-null mice (Takahashi et al. 2004). For neuroendocrine peptides, arginine-vasopressin gene expression is up-regulated in a compensatory manner in oxytocin knockout animals upon salt loading paradigms (Ozaki et al. 2004) and brain natriuretic peptides can compensate for atrial natriuretic peptide gene disruption in ventricular tissues (Tse et al. 2001). By contrast, knockout of the endothelin-1 gene does not result in upregulated expression of related endothelin transcripts
(Maemura et al. 1994) and bCGRP (b calcitonin gene-related peptide) transcripts are diminished from wild-type levels in dorsal root ganglia of aCGRP knockout mice (Gangula et al. 2000). Accordingly, the expression and responses of related genes in knockout studies can be quite varied. If compensatory mechanisms in the PACAP or VIP deletion models were a consideration, similar to the arginine vasopressin/oxytocin and brain natriuretic peptide/atrial natriuretic peptide compensatory relationships described above, then one expectation in the PACAP gene knockout might be an up-regulation in VIP expression beyond wildtype levels. Conversely, an increase in PACAP levels may be anticipated in the VIP knockouts. Our current comparative postnatal developmental studies are important from several vantages. Firstly, from the perspective of normal postnatal brain development, PACAP and VIP have vastly different temporal patterns of expression. PACAP peptide and transcript levels were already high at birth, which appears consistent and extends previous work demonstrating the prominent expression of PACAP during fetal nervous system development. These early PACAP expression patterns have been suggested to participate in neuro/gliogenesis, cell specification and differentiation. The developmental increase in PACAP expression continued postnatally and was 2-fold over birth levels by P7, which already approximated concentrations in adult brain. On a femtomole basis, brain VIP peptide levels at birth were lower than those for PACAP, which again was consistent with the negligible to low expression of VIP mRNA in fetal brain (Waschek et al. 1996). In contradistinction to postnatal PACAP expression patterns, VIP levels increased greater than 50-fold on a very protracted developmental scale spanning maturity. Secondly, compared to wild-type peptide levels, the PACAP or VIP knockout animals did not appear to present overt compensatory increases in the expression of the reciprocal peptide by radioimmunoassay or quantitative PCR measures of peptide content or transcripts, respectively. The developmental increases in PACAP expression in the VIP knockouts, for example, were apparently insufficient to compensate for the loss in VIP expression. The magnitude and temporal parameters for PACAP expression in the VIP knockouts was similar to patterns in wild-type animals. If PACAP were to compensate for VIP loss, then PACAP peptide levels in the VIP knockout animals may have been anticipated to mirror VIP developmental expression and exceed wild-type PACAP levels by 2- to 5-fold. Conversely, VIP expression in the PACAP knockouts appeared comparable to wild-type VIP patterns. Although these were more global measures of brain PACAP and VIP expression levels and may have overlooked potential compensatory peptidergic responses in restricted nuclei, PACAP and VIP are highly expressed in prominent CNS regions including cortex, hippocampus, hypothalamus and cerebellum and changes in peptide expression patterns in these structures are anticipated to be
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reflected in these analyses (Rosselin et al. 1982; Jaworski and Proctor 2000; Hannibal 2002). In contrast to the anticipated compensatory increases in reciprocal peptide expression, there was evidence for developmental delays or disruptions in both knockout strains. During crucial and sensitive periods of CNS development (environs P7) when neuronal maturation, including axonal growth, myelination, receptor/channel expression, and synapse formation, are developing into adult patterns, the expression of PACAP or VIP in the reciprocal knockouts was lower than wild-type levels of the same age. The decrements were evident from independent peptide content and transcript measurements to substantiate these changes, and appeared selective for PACAP and VIP as the expression of other peptides, such as galanin, appeared unaffected in the knockout tissues (data not shown). The mechanisms underlying the decreases in PACAP and VIP levels at this age are unclear but as PACAP and VIP can have trans- and autoregulatory functions (Mohney and Zigmond 1998; Girard et al. 2002), the diminution in brain peptide expression at P7 may reflect deficiencies in the reciprocal peptide. The developmental decrease in PACAP and VIP peptide levels in the knockouts was transient, as brain peptide content appeared to recover at later developmental stages. These recovery mechanisms are not understood but the increases in PACAP content may have resulted from enhanced peptide translation. Lastly, there were changes in PACAP and VPAC receptor subtype expression patterns during development. Whereas previous work describing the expression of PACAP and VIP receptor subtypes employed largely in situ hybridization approaches, the current studies provide more precise quantitative comparative measures. PAC1 and VPAC2 receptor transcript levels were already significant at birth and there were small or no apparent changes in expression levels during postnatal brain development, which appeared to be correlated with PACAP expression. Brain PACAP expression was already high at birth and in aggregate, the early expression of the PACAP, PAC1, and VPAC2 systems most likely reflects their prominent roles in development. By contrast, brain VPAC1 receptor transcript levels augmented approximately 10-fold from P1 through P28, which appeared to correlate closely with VIP expression in magnitude and temporal patterns. As for the peptides, no compensatory increases in receptor gene expression were observed in VIP or PACAP knockout mice. However, the present studies do not rule out the possibility that compensatory increases occurred at the level of receptor binding or signaling. The apparent lack of compensatory mechanisms between PACAP and VIP in the knockout animals suggests that despite the high degree of conservation in DNA sequence and structure within the gene, the promoter regions of PACAP and VIP have diverged considerably with respect to developmental regulation. The promoters for PACAP and
VIP genes have been described for some species and despite the presence of common regulatory regions, including cyclic AMP, cytokine and glucocorticoid responsive elements, the context of these elements within the promoters must be sufficiently different to alter the transcriptional expression of these peptide genes. The separate, apparently non-compensatory, properties of PACAP and VIP, and the resulting deficiencies in each peptide during critical periods of brain development may anticipate phenotypic differences between the two genotypes. Both previous and current studies have indicated that this is the case. Although the PACAP and VIP knockouts both display circadian rhythm defects, for example, the mechanisms underlying these phenotypes appear different. In the present work, VIP-deficient mice, like PACAP nulls, also display hyperactive behavior in transfer arousal assays; but unlike the PACAP knockouts with sustained hyperactivity, the increased hyperactivity in the VIP knockouts abated over time. This ability for PACAP knockout mice to sustain hyperactivity is singular and consistent with other reports on PACAP and PAC1 null mice behavior (Hashimoto et al. 2001; Otto et al. 2001b). Whether these responses reflect defects in dopaminergic motor control is unknown; alternatively PACAP or PAC1 receptor null mice may exhibit memory/learning deficits and/or an attention deficit hyperactivity-like phenotype. A potential action of PACAP/PAC1 signaling in learning and memory is suggested by the presence of PAC1 receptor in hippocampus and reports of alterations in LTP formation in PAC1 receptor deficient mice (Otto et al. 1999, 2001a; Matsuyama et al. 2003). In other neurological and behavioral assessments, PACAP knockouts appeared to exhibit less fear and anxiety, as shown by their tendency to traverse the open arena over time, and displayed defects in negative geotaxis behavior. The VIP null mice, by contrast, did not present the same responses. Although difficulties in performing negative geotaxis tasks are often related to abnormalities in vestibular/sensory systems, the PACAP null animals may have scored poorly because of their propensity to escape from the test apparatus. All 15 PACAP null animals in the trials were able to adequately perform the negative geotaxis task (turn and climb) at least once during repeated testing over the 1-month period. Together with their fearless behavior, these results suggest that PACAP null mice might have generalized reduced levels of fear. Previous reports implicating roles for VIP and PACAP in pain may have bearings on these observations in the PACAP knockout mice (Dickinson and Fleetwood-Walker 1999). Unlike PACAP knockout animals, mice with targeted VIP gene disruption displayed subtle but significant reductions in muscular strength as revealed in the grip strength and wire maneuver tasks. These results appear consistent with recent studies demonstrating that VIP and VPAC2 receptor signaling play key roles in the physiological control of skeletal
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muscle mass and ameliorating muscle loss from atrophic conditions (Hinkle et al. 2005). That this defect was confined to VIP knockout mice further indicates that endogenous PACAP does not compensate for VIP action on polyvalent VPAC2 receptors on skeletal muscle. In summary, these studies demonstrate that PACAP and VIP display differential expression patterns during postnatal mice brain development and are apparently non-compensatory peptidergic systems in reciprocal knockout genotypes. From these results, the distinct phenotypes of the PACAP or VIP deletion models most likely reflect disruption of corresponding genes rather than attenuated responses from related peptide expression. Acknowledgements This work was supported by HD27468 (VM/KMB), NS37179 (KMB/VM), DK065989 (MAV), HD34475 and HD06576 (JAW) and DC04736 (GVR) from the National Institutes of Health. The automated DNA sequencing and quantitative PCR were performed in the Vermont Cancer Center DNA analysis facility and were supported in part by grant P30CA22435 from the National Cancer Institute. The support of NIH grant P20RR16435 from the National Center for Research Resources (NCRR) is also gratefully acknowledged. The behavioral and functional assessments were performed at the UCLA Behavioral Testing Core established with funds from the Howard Hughes Medical Institute. We also wish to thank Alan Howard of the University of Vermont Computing and Information Technology Center for assistance in statistical analyses.
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