Optimization of long-term incubation of precision-cut kidney slices.

Precision-cut kidney slices (PCKS) provide a powerful model to close the gap between in vivo and in vitro research. Publications by various authors favor different incubation conditions, media, and antibiotics, that have not yet been compared in a standardized manner. After preparation, rat-PCKS were incubated in a total of nine combinations of incubation media and antibiotics for four days. We found that a combination of DMEM/F-12 and gentamicin showed the highest levels of viability. Utilizing both qualitative and quantitative methods, we observed stable levels of cellular viability for 10 days when incubated in the most suitable medium combination of DMEM and gentamicin. Additionally, a calcein acetoxymethyl/ethidium homodimer-1 based live/dead staining, analysis of total protein content and lactate dehydrogenase (LDH) were explored to assess both short- and long-term tissue viability. PCKS showed a significant decrease in total protein content, leveling off at around 60% over the duration of 10 days. To be able to evaluate viability irrespective of decreases in total protein detected, we chose to utilize the alamarBlue Cell Viability Assay. Quantifying both intra- and extracellular activity of LDH, while using different concentrations of ethanol as a positive control, we explored enzyme content as a parameter for cell membrane damage and cytotoxicity in PCKS. Overall, we showed that PCKS are suitable for both short- and long-term observation by optimizing incubation parameters, with numerous possibilities for other assays and methods in future studies.

Methicillin-resistant Staphylococcus aureus infection in combat support hospitals in three regions of Iraq.

SUMMARYStaphylococcus aureus is a leading cause of infections in deployed service members. Based on a molecular epidemiological study of 182 MRSA isolates from patients in three U.S. Army combat support hospitals in separate regions in Iraq, USA300 clone was the most predominant (80%) pulsotype. This finding suggested that strain carriage from the home country by military personnel is epidemiologically more important than local acquisition.

Molecular genetic analysis of 178 I-Abm12-reactive T cells.

We have studied the genetic diversity of the TCR repertoire to the murine alloantigen I-Abm12 by generating a panel of 178 C57BL/10-derived I-Abm12-reactive T cell hybridomas. The expression of V alpha and V beta gene families was examined in this panel and the frequency of expression of V beta, but not ofV alpha, gene families differed significantly from that observed in a companion panel of random C57BL/10-derived hybridomas. The V beta 5 gene family was expressed significantly less frequently while the V beta 14, V beta 15, and V beta 16 genes were expressed significantly more frequently in the panel of I-Abm12-reactive than in the panel of random hybridomas. The junctional regions (VJ alpha and VDJ beta) of TCR V alpha and V beta genes from selected I-Abm12-specific hybridomas were amplified using the polymerase chain reaction, and directly sequenced. Surprisingly, no conserved J alpha, D beta, J beta, or N region-encoded sequences among these selected I-Abm12-reactive TCRs were identified. Thus, the T cell response to an I-A alloantigen that differs by only three amino acid residues from the I-A molecule of the responding strain is genetically complex but nonrandom. We have estimated that the repertoire to this alloantigen is comprised of at least 37 different TCRs.

Imprinting: lasting effects on uracil incorporation into chick brain.

On the first day after hatching, domestic chicks were trained for 20, 60, 120, or 240 minutes with an imprinting stimulus. On the second day, they were all retrained for 60 minutes. The greater the chicks' experience on the first day, the lower the rate of incorporation of tritiated uracil into macromolecules in the anterior part of the forebrain roof on the second day. Such effects were not found in other brain regions, nor in any brain region of chicks that received similar treatment on the first day but were not retrained on the second.

Direct cloning and sequence analysis of enzymatically amplified genomic sequences.

A method is described for directly cloning enzymatically amplified segments of genomic DNA into an M13 vector for sequence analysis. A 110-base pair fragment of the human beta-globin gene and a 242-base pair fragment of the human leukocyte antigen DQ alpha locus were amplified by the polymerase chain reaction method, a procedure based on repeated cycles of denaturation, primer annealing, and extension by DNA polymerase I. Oligonucleotide primers with restriction endonuclease sites added to their 5' ends were used to facilitate the cloning of the amplified DNA. The analysis of cloned products allowed the quantitative evaluation of the amplification method's specificity and fidelity. Given the low frequency of sequence errors observed, this approach promises to be a rapid method for obtaining reliable genomic sequences from nanogram amounts of DNA.

Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.

Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.

A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.

[Gluteoplasty with intramuscular silicone cohesive gel implants: a retrospective study of 50 cases]. / Augmentation fessière par implants intramusculaires en gel cohésif de silicone : étude rétrospective de 50 cas consécutifs.

For a few years, surgery of the gluteus has become more and more popular. In this retrospective study of 50 cases over a period of five years, the author presents his results, using a personal technique of gluteoplasty with intramuscular implants. After a review of the different current surgical techniques and of the different types of gluteus, the author describes his own surgical strategy.

Visual imprinting and the neural mechanisms of recognition memory.

To understand the neural bases of memory it is necessary to localize the regions storing information. Part of the hyperstriatum ventrale (IMHV) serves such a function for the learning process of imprinting in domestic chicks. Chicks exposed to an object learn its characteristics, and in doing so, the responsiveness of IMHV neurones to that object is selectively enhanced. Imprinting is associated with both pre- and postsynaptic changes in the region. Postsynaptic changes involve increases in the length of the postsynaptic density on dendritic spines and in the numbers of NMDA receptors; presynaptically, converging evidence points to an early and persistent enhancement of neurotransmitter release. Increases in the amounts of certain neural cell adhesion molecules a day after training might serve to stabilize the synaptic changes associated with a particular memory by strengthening pre- to postsynaptic adhesion, and by more strongly interconnecting the cytoskeletal frameworks of the dendritic spine and the synaptic terminal. Learning-related increases in the number of neurones staining positive for the transcription factor Fos in the IMHV give promise of identifying the neurones engaged in memory functions and of analysing their connections.

Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N).

A catalytically active fragment of the Rap-specific guanine-nucleotide exchange factor C3G was expressed in E coli. It was purified and its interaction with GTP-binding proteins was investigated using fluorescence spectroscopy. C3G stimulates GDP dissociation from Rap1, but not from Rap2, neither from Bud1, which is believed to be the yeast homologue of Rap1 nor from all other proteins of the human Ras-subfamily. Like the corresponding fragment from CDC25Mm, the increase in the GDP dissociation rate is linear with increasing concentration of Rap1A x GDP up to 100 microM, indicating an apparent K(M) higher than 100 microM. Unlike the Ras-CDC25Mm system, the Rap1A(S17N) mutant does not inhibit the C3G-activated guanine nucleotide dissociation from wild-type Rap1A in vitro. These data suggest that Rap1A(S17N) is unlikely to titrate away C3G in vivo, the proposed mechanism by which S17N-mutants exert their dominant negative effects.

Tyrosine hydrogen bonds make a large contribution to protein stability.

The aim of this study was to gain a better understanding of the contribution of hydrogen bonds by tyrosine -OH groups to protein stability. The amino acid sequences of RNases Sa and Sa3 are 69 % identical and each contains eight Tyr residues with seven at equivalent structural positions. We have measured the stability of the 16 tyrosine to phenylalanine mutants. For two equivalent mutants, the stability increases by 0.3 kcal/mol (RNase Sa Y30F) and 0.5 kcal/mol (RNase Sa3 Y33F) (1 kcal=4.184 kJ). For all of the other mutants, the stability decreases with the greatest decrease being 3.6 kcal/mol for RNase Sa Y52F. Seven of the 16 tyrosine residues form intramolecular hydrogen bonds and the average decrease in stability for these is 2.0(+/-1.0) kcal/mol. For the nine tyrosine residues that do not form intramolecular hydrogen bonds, the average decrease in stability is 0.4(+/-0.6) kcal/mol. Thus, most tyrosine -OH groups contribute favorably to protein stability even if they do not form intramolecular hydrogen bonds. Generally, the stability changes for equivalent positions in the two proteins are remarkably similar. Crystal structures were determined for two of the tyrosine to phenylalanine mutants of RNase Sa: Y80F (1.2 A), and Y86F (1.7 A). The structures are very similar to that of wild-type RNase Sa, and the hydrogen bonding partners of the tyrosine residues always form intermolecular hydrogen bonds to water in the mutants. These results provide further evidence that the hydrogen bonding and van der Waals interactions of polar groups in the tightly packed interior of folded proteins are more favorable than similar interactions with water in the unfolded protein, and that polar group burial makes a substantial contribution to protein stability.

Technical note: A method for assigning animals to treatment groups with unequal count per group that equalizes mean animal weight among groups.

Pastures available for grazing studies may be of unequal size and may have heterogeneous carrying capacity necessitating the assignment of unequal numbers of animals per pasture. To reduce experimental error, it is often desirable that the initial mean BW be similar among experimental units. The objective of this note is to present and illustrate the use of a method for assignment of animals to experimental units of different sizes such that the initial mean weight of animals in each unit is approximately the same as the overall mean. Two alternative models were developed and solved to assign each of 231 weaned steers () to 1 of 12 pastures with carrying capacity ranging from 5 to 26 animals per pasture. A solution to Model 1 was obtained in which the mean weights among pastures were approximately the same but the variances among pastures were heteroskedastic, meaning that weight variances across pens were different (-value < 0.05). An alternative model was developed (Model 2) and used to derive assignments with nearly equal mean weights and homoskedastic variances among pastures.

Coordinated gene expression between skeletal muscle and intramuscular adipose tissue in growing beef cattle.

Previous research indicates that metabolism and fiber type of skeletal muscle is related to intramuscular lipid content. It is hypothesized that changes in skeletal muscle gene expression influence adipose tissue development. The objective of this study was to determine differences in the metabolism and intercellular signaling of skeletal muscle fibers within the same muscle group that could be responsible for the initiation of intramuscular adipose tissue development and differentiation. Longissimus dorsi muscle samples were collected from steers ( = 12; 385 d of age; 378 kg BW) grazing wheat pasture. Longissimus muscle samples were dissected under magnification and sorted into 3 categories based on visual stage of adipose tissue development: immature intramuscular adipose tissue (MM), intermediate intramuscular adipose tissue (ME), and mature intramuscular adipose tissue (MA). Additionally, muscle fibers lying adjacent to each intramuscular adipose tissue (IM) category and those not associated with IM tissue were collected and stored separately. Quantitative real-time PCR was used to determine relative fold change in genes involved in metabolism, angiogenesis, formation of extracellular matrix, and intercellular signaling pathways in both LM and IM samples. Gene expression data were analyzed using a GLM that included the fixed effect of tissue. Pearson correlation coefficients were also computed between gene expression in LM and IM tissue samples that were at the same stage of development. and γ mRNA expression were 3.56- and 1.97-fold greater ( < 0.05) in ME and MA IM compared with MM IM whereas mRNA expression was 1.43-fold less ( < 0.01) in MA IM compared with MM IM, indicating successful separation into different development categories. Genes associated with metabolism and angiogenesis in LM tissue showed no differences among stages of development. Myostatin expression did not change in LM tissue; however, expression of and mRNA decreased ( < 0.01) as IM matured. and mRNA expression were 2.5- and 1.32-fold greater in LM associated with MM IM than in LM associated with ME IM. Angiogenic growth factors in MM IM tissue had a strong positive correlation ( ≥ 0.69) with angiogenic growth factors in LM associated with MM IM; however, no correlation was observed in ME or MA IM. These data indicate a coordinated effort between LM and IM in early stages of IM development.

In vivo ruminal degradation characteristics and apparent digestibility of low-quality prairie hay for steers consuming monensin and Optimase.

Seven ruminally cannulated crossbred steers (BW = 720 ± 62 kg) were used in a randomized crossover design (4 periods, each 18 d) to evaluate in vivo rumen characteristics and apparent digestibility of steers consuming low-quality prairie hay and 1 of 4 isonitrogenous protein supplements. Treatments included 1) 40% CP (DM basis) cottonseed meal and wheat middlings-based supplement (Control), 2) a cottonseed meal and wheat middlings-based supplement with slow-release urea and a fibrolytic feed enzyme (Optimase; Alltech, Inc., Nicholasville, KY) designed to replace 30% of plant-based CP provided in the Control (OPT), 3) the Control plus 0.40 mg∙kg BW∙d monensin (Rumensin 90; Elanco Animal Health, Greenfield, IN; MON), and 4) the OPT plus 0.40 mg∙kg BW∙d monensin (COMBO). Steers were allowed ad libitum access to prairie hay (5.0% CP and 76% NDF) and were provided each respective supplement at 0800 h daily at a rate of 1.0 g/kg of BW. Steers were adapted to diets for 10 d before sample collection. Beginning on d 11, DMI was measured and samples were collected to determine apparent digestibility. On d 15 of the 18-d period, rumen fluid was collected 10 times over a 24-h period. Forage DMI was greater ( ≤ 0.02) for steers consuming the OPT compared with steers consuming the MON or COMBO, although forage DMI was not different ( = 0.10) among steers consuming the Control compared with steers consuming the OPT, MON, or COMBO. Steers fed the MON and COMBO had lower ( ≤ 0.05) passage rate compared with steers fed the Control and the OPT. The MON-fed steers had lower ( = 0.01) ruminal pH and increased ( = 0.03) propionate as a percentage of total VFA production. A time × treatment ( = 0.01) interaction was observed for ruminal NH-N due to a rapid (0 to 1 h after feeding) increase followed by a quick (1 to 4 h after feeding) decline in NH-N by steers consuming the OPT and COMBO that was not observed for steers consuming all other treatments. Apparent digestibility of DM ( = 0.01) and NDF ( = 0.03) were improved for steers fed the COMBO supplement compared with steers consuming all other experimental supplements. This work suggests that the OPT may be an effective replacement for a portion of supplemental degradable intake protein in low-quality forage. Further research is necessary to determine if the combination of monensin and the Optimase consistently improves low-quality forage utilization.

Effect of rate of weight gain of steers during the stocker phase. IV. Rumen fermentation characteristics and expression of genes involved in substrate utilization for fatty acid synthesis in adipose tissues of growing-finishing beef cattle.

The objective of this study was to determine the impact of stocker production systems differing in growth rate on rumen fermentation characteristics and utilization of substrates for fatty acid synthesis in intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissues. Angus steers were assigned to 4 stocker cattle production systems in 2 consecutive years: 1) 1.0 kg/d of 40% CP cottonseed meal­based supplement while grazing dormant native range (CON), 2) ground corn/soybean meal­based supplement while grazing dormant native range fed at 1% of BW (CORN), 3) grazing wheat pasture at a high stocking rate to achieve a low rate of BW gain (LGWP), and 4) grazing wheat pasture at a low stocking rate for a high rate of BW gain (HGWP). Eight ruminally cannulated steers were used to determine rumen fermentation characteristics. Steers were harvested during the stocker phase at similar age (different carcass weight) in Exp. 1 (3 steers/treatment) or at similar carcass weight in Exp. 2 (4 steers/treatment). Adipose tissues were analyzed for mRNA expression of genes involved in glucose (solute carrier family 2, member 4 [GLUT4], glucose-6-phosphate dehydrogenase [G6PDH], phosphofructokinase, muscle [PFKM], and pyruvate kinase 2, muscle [PK2]), lactate (lactate dehydrogenase B [LDHB]), and acetate (acetyl-CoA synthetase, cytosol [ACSS2]) utilization for fatty acid synthesis. The acetate:propionate ratio was least (P < 0.05) for HGWP steers, intermediate for CORN and LGWP steers, and greatest for CON steers. At similar age, LGWP and HGWP steers tended (F-test; P < 0.15) to have greater (P < 0.10) G6PDH and ACSS2 mRNA expression than CON and CORN steers in SC and PR but not IM adipose tissue. Expression of PFKM and PK2 mRNA tended (F-test; P < 0.15) to be greater (P < 0.10) in HGWP than CON and LGWP steers in IM but not SC or PR adipose tissue. At similar HCW, expression of GLUT4 and G6PDH mRNA were greater (P < 0.10) in SC adipose tissue of LGWP and HGWP steers compared with CON and CORN steers but not in IM and PR adipose tissue. Expression of LDHB mRNA was lesser (P < 0.10) in SC adipose tissue but greater (P < 0.10) in PR adipose tissue of LGWP and HGWP steers compared with CON and CORN steers. These results indicate a shift toward glucose utilization in SC adipose tissue but a shift towards lactate utilization in PR adipose tissue. These results suggest that diet and changes in VFA profile can influence substrates utilized for fatty acid synthesis, but diet has a greater effect in SC than IM adipose tissue.

Supplementation of monensin and Optimase to beef cows consuming low-quality forage during late gestation and early lactation.

Two experiments were designed to investigate the effects of feeding monensin and/or slow release urea with a fibrolytic feed enzyme (Optimase; Alltech, Inc., Nicholasville, KY) on performance, milk production, calf growth performance, and blood metabolites in beef cows. Spring-calving cows and heifers were used in a completely randomized design in Exp. 1 (N = 84; 534 ± 68 kg initial BW) and Exp. 2 (N = 107; 508 ± 72 kg initial BW). Exp. 1 supplements were formulated to meet cow protein requirements and fed daily and included 1) cottonseed meal with no monensin (control); or 2) monensin added to control to supply 200 mg per head per d (MON). In Exp. 2, experimental supplements included 1) cottonseed meal/wheat middlings (CS) fed at a rate to provide adequate DIP and CP according to , 2) the CS plus soybean hulls and 61 g per cow per d Optimase (OPT), 3) the CS plus monensin to supply 200 mg per cow per d (MON2), and 4) OPT plus MON2 (Combo). Cows were fed in last trimester through early lactation in Exp. 1 and during 2nd trimester in Exp. 2. Data were analyzed using the Mixed procedure in SAS with animal as the experimental unit. In Exp. 1, treatment did not affect cow BW or BCS change (P > 0.19). Calf birth BW was not affected by dam treatment (P = 0.24); however, calves from dams consuming MON weighed more (P < 0.04) at d 45 and at trial end. Calves also had greater (P = 0.04) ADG from birth to trial end. Milk production did not significantly differ among treatments (P > 0.41). In Exp. 2, mean cow BW and BCS were similar (P > 0.35) among treatments on d 90. However, from d 0 to 54, cows assigned to the OPT supplement gained less BCS (P = 0.02) compared with cows assigned to the CS supplement. Cumulative BCS gain was greater (P < 0.01) for CS-fed cows than for cows fed the OPT and MON2 supplements, although it was not significantly different for cows fed the Combo supplement. These studies indicate that the influence of monensin on cow BW and BCS change is inconsistent. The potential for monensin supplementation to positively impact calf performance during early lactation seems to be clearer. Replacing a portion of oilseed N in the supplement with Optimase may marginally reduce cow performance. Further research is needed to determine both the effects of monensin and the implications of combining monensin with Optimase on forage intake and cow performance at various stages of production.

Serological and polymerase chain reaction-based analysis of aqueous humour samples in patients with AIDS and necrotizing retinitis.

OBJECTIVE: To evaluate the measurement of intraocular antibody production and detection of DNA by the polymerase chain reaction (PCR) for diagnosis of the causative microorganism in patients with AIDS and necrotizing retinitis. METHODS: Paired serum and aqueous humour samples obtained from 28 patients with AIDS and necrotizing retinitis, seen between January 1987 and March 1992, were analysed for intraocular antibody production against cytomegalovirus (CMV), varicella zoster virus, herpes simplex virus, Epstein-Barr virus, and Toxoplasma gondii. Specific antibody titres in the inflamed eye and in the circulation were related to total immunoglobulin G content in the aqueous humour and serum. In addition, PCR analysis was performed in 15 samples. Results were compared to the final diagnosis, which was based on the subsequent clinical course. Results were also related to parameters describing the immune state of the patients: CD4 count, time between diagnosis of an AIDS-defining illness and retinitis, and time of survival following the diagnosis of retinitis. RESULTS: In 11 (39%) out of 28 patients we found local intraocular antibody production which correlated with the final diagnosis (one out of two cases with acute retinal necrosis, three out of five cases with toxoplasma retinitis, and eight out of 21 patients with CMV retinitis). In all 13 patients with CMV retinitis PCR analysis detected CMV DNA. In one patient with the clinical diagnosis of Toxoplasma retinitis, Toxoplasma DNA could be determined, whereas in the same sample CMV DNA was also found. In yet another patient with Toxoplasma retinitis only CMV DNA could be detected. A relationship between results of local antibody determination with either CD4 counts, or the time interval between AIDS-defining illness and retinitis, or survival time after diagnosis of retinitis could not be established. CD4 counts were higher than 50 x 10(6)/l in eight out of 19 patients with CMV retinitis. No complications of paracentesis were seen. CONCLUSIONS: Detection of intraocular antibody production and PCR analysis are quick and safe procedures and helpful tools for diagnosis of the involved pathogen in AIDS patients with a necrotizing retinitis. Negative results of local antibody production, even in the presence of detectable viral DNA, could not be related to the parameters of a more deteriorated immune status of these patients.

Cytomegalovirus (CMV) strain differences between the eye and blood in AIDS patients with CMV retinitis.

OBJECTIVE: To investigate possible differences in cytomegalovirus (CMV) strain distribution between the eye and blood in AIDS patients with CMV retinitis. METHODS: CMV DNA sequences from aqueous humour and peripheral blood leukocytes (PBL), obtained from 13 AIDS patients with CMV retinitis, were compared. DNA was isolated and the CMV IE-1 sequence (part of the immediate early-1 gene) and the a-sequence (located in the a-region) were amplified by polymerase chain reaction (PCR). The PCR products of the a-sequence were analysed by Southern blotting for amplified fragment-length polymorphisms. The level of divergence between the a-sequences of aqueous humour- and PBL-derived CMV was studied in two patients by cloning these sequences followed by sequence analysis. RESULTS: CMV DNA could be detected in all aqueous humour samples and in 10 out of 13 paired blood samples. In the 10 patients, with CMV DNA detectable in both aqueous humour and PBL, seven cases showed differences between the amplified products of both compartments. Sequence analysis in two patients revealed that the aqueous humour and PBL of the same patient can harbour both identical, similar and highly divergent CMV a-sequences. CONCLUSION: These results indicate that despite the haematogenous spread of CMV, the eye, being a relatively shielded organ, may contain CMV strains different from those found in the blood.

High-pressure NMR study of the complex of a GTPase Rap1A with its effector RalGDS. A conformational switch in RalGDS revealed from non-linear pressure shifts.

Unusually large non-linear 1H and 15N nuclear magnetic resonance chemical shifts against pressure have been detected for individual amide groups of the Ras-binding domain of Ral guanine dissociation stimulator (GDS). The non-linear response is largest in the region of the protein remote from the Rap1A-binding site, which increases by about two-fold by the complex formation with its effector protein Rap1A. The unusual non-linearity is explained by the increasing population of another conformer (N'), lying energetically above the basic native conformer (N), at higher pressure. It is considered likely that the conformational change from N to N' in the Ras-binding domain of RalGDS works as a switch to transmit the effector signal further to molecules of different RalGDS-dependent signaling pathways.

Learning-dependent Changes in the Responses to Visual Stimuli of Neurons in a Recognition Memory System.

When young chicks are trained by exposing them to a conspicuous object they learn its characteristics. The learning process is known as imprinting. In the present study neuronal activity in a region crucial for imprinting was shown to be affected by training and by the object on which the chicks had been trained. The region is the intermediate and medial part of the left hyperstriatum ventrale (left IMHV). No such effects were found in a visual projection area, the left hyperstriatum accessorium. Domestic chicks were imprinted on either a rotating red box (n=7 chicks) or a rotating blue box (n=8). When the chicks were approximately 48 h old they were anaesthetized and multiple-unit activity was recorded in simultaneous, single penetrations through each of the two regions. Records were also made from eight dark-reared chicks. Whilst recording, the red or blue box, placed in front of the contralateral eye, was switched on to give a total of 20 rotations, the interval between each rotation being 10 s. The alternative stimulus was then presented 20 times. Unit activity in the 3 s before and after stimulus onset was compared and the data for each of the 20 presentations were combined. In the left IMHV 18 out of a total of 115 recording sites (16%) responded significantly to the stimuli; in the left hyperstriatum accessorium 39 out of 126 recording sites (26%) did so. Measures of unit activity at each recording site were combined for a given penetration to provide a 'mean penetration response'. The response to the red box differed from the response to the blue box in the left IMHV of dark-reared chicks. After training with the blue box the response to both boxes was similar to the response to the blue box in dark-reared birds. After training with the red box the response to both boxes was similar to the response to the red box in dark-reared birds. No significant effects were found in the left hyperstriatum accessorium. The two training boxes were virtually identical apart from the differences in colour and brightness. Training appeared to stabilize the response of the visually naive left IMHV to the training stimulus whilst changing its response to the alternative, but similar stimulus. That is, one consequence of training is that the two stimuli are placed in the same category, and this neural change may provide a basis for stimulus generalization. The underlying neural system is modelled and a mechanism that allows such stimuli to be discriminated is proposed.